Recombinant fusion protein and polynucleotide construct for immunotoxin production

ABSTRACT

The present invention relates to a polynucleotide construct encoding a fusion protein consisting of a domain which binds the immunoglobulin Fc region, genetically fused to a truncated form of  Pseudomonas  exotoxin A (PE). In particular, the invention discloses the fusion protein, ZZ-PE38, and further provides immunotoxins, formed from complexes of the fusion protein with antibodies for targeted cell killing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.12/117,494 filed May 8, 2008, which application claims the benefit ofapplication No. 60/917,160 filed May 10, 2007. The entire content ofeach application is expressly incorporated herein by reference thereto.

FIELD OF THE INVENTION

The present invention relates to a polynucleotide construct encoding afusion protein consisting of a domain which binds the immunoglobulin Fcregion, genetically fused to a truncated form of Pseudomonas exotoxin A(PE). The present invention further provides immunotoxins, formed fromcomplexes of the fusion protein with antibodies for targeted cellkilling.

BACKGROUND OF THE INVENTION

The aim of drug targeting is to kill target cells such as cancer cells,while leaving normal tissues unharmed. Immunotoxins combine theselectivity of an antibody moiety with the potency of a toxin moiety.Such agents kill target cells via a process which involves specificbinding to a cell surface antigen preferentially expressed on the cells,such as a tumor-associated antigen, internalization and delivery of thetoxic moiety to the cytosol, where a critical cell function isinhibited, leading to cell death. A decisive breakthrough in immunotoxindevelopment was the advent of hybridoma technology, making monoclonalantibodies (mAbs) available in limitless supply. The “first generation”of immunotoxins, for example as disclosed in U.S. Pat. No. 4,545,985,were chemically linked conjugates of mAbs or Fab′ fragments capable ofbinding cancer cell antigens, and potent protein toxins derived fromplants or bacteria such as ricin, abrin, saporin, Pseudomonas aeruginosaexotoxin (PE), cholera toxin (CT) and Diphtheria toxin (DT). Such earlyimmunotoxins showed impressive results in vitro but in most casesdisplayed poor anti-tumor effects in animals or humans, and often withexcessive toxicity.

The “second generation” of immunotoxins were generally fully recombinantantibody-toxin chimeric molecules, usually in the form of a single-chainantibody genetically fused to a truncated version of either DT or PE,such as disclosed for example in U.S. Pat. No. 6,051,405.

Over the years, a large number of antibodies that bind tumor-associatedantigens have been isolated. Early on, the need for a rapid screeningapproach to assess the potential of such antibodies was recognized,since internalization is a pre-requisite for most drug deliveryapproaches (Casalini et al 1993, Cancer Immunol Immunother 37, 54-60).An undisputed proof of internalization can be provided by linking theantibody to a cytotoxic cargo (such as a drug or a toxin) and testingthe ability of the antibody to deliver the cargo into a target cell. Thefirst generation of immunotoxins could provide such a tool, but someantibodies are not readily conjugated. While use of second generationrecombinant immunotoxins for screening purposes is technically feasible,it is extremely labor intensive.

Some agents that could potentially link any IgG to a toxin have beendisclosed, for example in Kim and Weaver 1998, Gene 68, 315-21; O'Hareet al 1990, FEBS Lett 273, 200-4; Madshus et al 1991, J Biol Chem 266,17446-53; Tonevitskii et al 1991, Mol Biol (Mosk) 25, 1188-96, but noneof these disclosures show an agent effective in target cell killing.

The immunoglobulin Fc-binding protein denoted ZZ is a recombinant tandemrepeated, mutated form of domain B of protein A from Staphylococcusaureus which has been used in a variety of biotechnological applications(Nilsson et al 1987, Protein Eng. 1, 107-13; Nilsson et al 1996, ProteinEng. 1, 107-13).

Fusion proteins composed of protein ZZ and diphtheria toxin, either thefull-length toxin or fragment B thereof, have been disclosed (Madshus etal 1991, supra; Nizard et al 1998, FEBS Lett 433, 83-8). A chimericprotein composed of S. aureus protein A fragments and Pseudomonasaeruginosa exotoxin A has been disclosed (Tonevitskii et al 1991,supra). While the chimeric protein was shown to be capable ofADP-ribosylation of elongation factor 2 and binding to immunoglobulin,evaluation of its cytotoxic properties in two model systems showed onlya slight inhibition of target cell growth.

U.S. Pat. No. 5,917,026 discloses DNA sequences encoding fusion proteinscomprising a first segment which encodes a native or mutant subunit of abacterial toxin that confers enzymatic ADP-ribosylating activity interalia Pseudomonas toxin, and a second segment which encodes a peptidewhich confers water solubility on the fusion protein and targets thefusion protein to a specific cell receptor different from receptorsbinding to the native toxin, and can thereby mediate intracellularuptake of at least the toxin subunit. According to the disclosure, thereceptor may be one present on B lymphocytes and the peptide may beinter alia S. aureus protein A or a fragment thereof in single ormultiple copies. The only fusion proteins specifically disclosed arethose composed of cholera toxin subunit A1 linked to DD, the latterbeing a dimer of the D-region of protein A, and such fusions aredescribed as being non-toxic in vivo and capable of enhancing immuneeffects of B and T cells. According to the disclosure, the intended useof the fusion protein is for potentiating immune responses.

There remains a need for effective immunotoxins and recombinant reagentsfor screening of antibodies for their potential as components of suchimmunotoxins, in particular the ability to be internalized within targetcells.

SUMMARY OF THE INVENTION

The present invention provides for the first time a recombinant fusionprotein denoted as ZZ-PE, which comprises the IgG Fc-binding ZZ domainderived from S. aureus protein A (ZZ) genetically fused to a specifictruncated form of Pseudomonas aeruginosa exotoxin A (PE), and apolynucleotide construct encoding same. The ZZ-PE forms surprisinglytight complexes with IgG and other proteins comprising an Fc-regionderived from IgG. When complexed with IgG, the ZZ-PE does not underminespecific antigen recognition. The inventors have surprisingly found thatthe ZZ-PE fusion protein can be used as a “adaptive” reagent forconverting internalizing IgG antibodies into immunotoxin complexes orconjugates for targeted cell killing. More particularly, complexes orconjugates comprising the ZZ-PE fusion protein and IgG antibodies whichare efficiently internalized into target cells expressing the antigenrecognized by the antibody, can be used as pharmaceutical reagents fortargeted cell killing, for example of cancer cells.

Moreover, the present invention provides a composition comprising therecombinant fusion protein ZZ-PE and an anti-ErbB-1 antibody. Theinventors have found that the ZZ-PE fusion protein can be used togetheran anti-ErbB-1 antibody as immunotoxin complexes or conjugates fortargeted ErbB-1 expressing cell killing. These complexes or conjugatesare efficiently internalized into target cells expressing ErbB-1 and canbe used as pharmaceutical reagents for targeted cell killing, forexample of cancer cells.

According to a first aspect, the present invention provides apolynucleotide construct encoding a fusion protein, wherein theconstruct comprises a first nucleotide sequence encoding animmunoglobulin Fc-binding domain and a second nucleotide sequenceencoding a truncated form of Pseudomonas exotoxin.

As used herein, an “immunoglobulin Fc-binding domain” refers to aprotein which binds the Fc region of immunoglobulin molecules.

According to one embodiment, the immunoglobulin Fc-binding domain isderived from S. aureus protein A. According to one embodiment, theimmunoglobulin Fc-binding domain derived from S. aureus protein Acomprises domain B of protein A. According to one embodiment, the firstnucleotide sequence encoding the immunoglobulin Fc-binding domain is insingle or multiple copies. According to one embodiment, theimmunoglobulin Fc-binding domain derived from S. aureus protein A isdenoted as ZZ and has the amino acid sequence of SEQ ID NO: 2. Accordingto one embodiment, the immunoglobulin Fc-binding domain is an anti-Fcsingle chain antibody.

According to one embodiment, the truncated form of Pseudomonas exotoxinhas an amino acid sequence selected from the group consisting of SEQ IDNO:3 (denoted as PE38); SEQ ID NO:23 (denoted as PE38KDEL), SEQ ID NO:24(denoted as PE38RDEL), SEQ ID NO:25 (denoted as PE37) and SEQ ID NO:26(denoted as PE40).

According to a currently preferred embodiment the truncated form ofPseudomonas exotoxin is that denoted as PE38 and has the amino acidsequence of SEQ ID NO:3.

According to one embodiment, the first nucleotide sequence and thenucleotide second sequence of the polynucleotide construct are joined bya linker nucleotide sequence, wherein the linker nucleotide sequenceencodes a peptide linker having from four to 20 amino acids. Accordingto one embodiment, the peptide linker comprises SEQ ID NO:27. Accordingto one embodiment, the polynucleotide construct encodes a fusion proteinthat has the amino acid sequence of SEQ ID NO:1.

According to another embodiment, the polynucleotide comprises thesequence of SEQ ID NO:6. According to another embodiment, an expressionvector comprises the polynucleotide construct of the invention.According to one embodiment, the expression vector comprises a T7promoter and a pelB leader for secretion operably linked to thepolynucleotide. According to another embodiment, the expression vectorcomprises the sequence of SEQ ID NO:5.

According to another embodiment, the expression vector has the sequenceof SEQ ID NO:5.

According to yet another embodiment, a host cell comprises thepolynucleotide construct of the invention.

According to one embodiment, the host cell is selected from the groupconsisting of a prokaryotic cell and a eucaryotic cell. According to oneembodiment, the prokaryotic cell is a bacterium. According to oneembodiment, the bacterium is from a strain of Escherichia coli.

According to one embodiment, the host cell is selected from the groupconsisting of a prokaryotic cell and a eucaryotic cell. According to oneembodiment, the cell expressed ErbB-1.

According to another aspect, the present invention provides arecombinant fusion protein, wherein the protein comprises a firstsegment which is an immunoglobulin Fc-binding domain, and a secondsegment which is a truncated form of Pseudomonas exotoxin.

In one embodiment, the Fc-binding domain is derived from S. aureusprotein A. In one embodiment, the Fc-binding domain derived from S.aureus protein A comprises domain B of protein A. In one embodiment, theFc-binding domain derived from S. aureus protein A is denoted as ZZ andhas the amino acid sequence of SEQ ID NO: 2. According to oneembodiment, the immunoglobulin Fc-binding domain is present in thefusion protein in single or multiple copies. According to oneembodiment, the immunoglobulin Fc-binding domain is an anti-Fc singlechain antibody.

In another embodiment, the truncated form of Pseudomonas exotoxin has anamino acid sequence selected from the group consisting of SEQ ID NO:3(denoted as PE38); SEQ ID NO:23 (denoted as PE38KDEL), SEQ ID NO:24(denoted as PE38RDEL), SEQ ID NO:25 (denoted as PE37) and SEQ ID NO:26(denoted as PE40).

According to one embodiment the truncated form of Pseudomonas exotoxinis that denoted as PE38 and has the amino acid sequence of SEQ ID NO:3.

According to one embodiment, the first segment and the second segment ofthe fusion protein are joined by a peptide linker having from four to 20amino acids. According to one embodiment, the peptide linker has thesequence of SEQ ID NO:27.

According to a currently preferred embodiment, the fusion protein hasthe amino acid sequence of SEQ ID NO: 1. The fusion protein having theamino acid sequence of SEQ ID NO:1 is specifically referred to herein as“ZZ-PE38”.

According to one embodiment, the fusion protein is encoded by thesequence of SEQ ID NO:6. According to one embodiment, the fusion proteinis expressed by the expression vector having the sequence of SEQ IDNO:5.

According to one embodiment, an immunotoxin comprises a complex of thefusion protein of the invention and an antibody, wherein the antibody iscapable of specifically binding expressed on the surface of a targetcell. In one embodiment, the antibody is selected form the groupconsisting of a monoclonal antibody, a humanized antibody, a chimericantibody, a single chain antibody, and a fragment thereof. In oneembodiment, the antibody is capable of being internalized into thetarget cell. According to one embodiment, the antibody is an IgG1.

According to one embodiment, the immunotoxin comprises a fusion protein,wherein the fusion protein has the amino acid sequence of SEQ ID NO:1and an anti-ErbB-1 antibody.

In one embodiment, the complex is an immuno complex. In one embodiment,the complex is a chemically conjugated complex. In one embodiment, thecomplex is a cross-linked complex. In one embodiment, the target cell isselected from the group consisting of a cancer cell and a pathogeninfected cell. In one embodiment, the pathogen is selected from thegroup consisting of a parasite and a virus.

According to another aspect, the antigen is selected from the groupconsisting of a tumor-associated antigen, a pathogen-associated antigen,a parasite-associated antigen and a virus-associated antigen. In oneembodiment, the tumor-associated antigen is selected from the groupconsisting of MUC1 and ErbB2. In one embodiment, the target cell is acancer cell. In one embodiment, the cancer cell is from a site selectedfrom the group consisting of lung, colon, rectum, breast, ovary,prostate gland, head, neck, bone, kidney, liver, skin and the immunesystem. In one embodiment, the cancer cell is a breast cancer cell.

In one embodiment, the invention provides a method for selectivelykilling a target cell expressing ErbB-1 in a subject in need thereof,the method comprising the steps of administering to the subject aneffective amount of the composition comprising the anti-ErbB-1 antibodycomplexed to the fusion protein. According to another aspect, theinvention provides a method for selectively killing a target cell in asubject in need thereof, the method comprising the steps of:

-   -   a. providing a recombinant fusion protein, wherein the fusion        protein comprises a first segment which is an immunoglobulin        Fc-binding domain and a second segment which is a truncated form        of Pseudomonas exotoxin;    -   b. selecting an antibody which is specific for an antigen        present on the target cell and which is capable of being        internalized into the target cell;    -   c. combining the antibody from (b) with the fusion protein        from (a) so as to form an immunotoxin complex; and    -   d. exposing the target cells to an effective amount of the        immunotoxin complex of (c) under conditions which enable        internalization of the immunotoxin complex, thereby selectively        killing the target cell in the subject in need thereof.

According to another aspect, the invention provides a method forselectively killing a target cell over-expressing ErbB-1 in a subject inneed thereof, the method comprising the steps of administering to thesubject an effective amount of the composition comprising theanti-ErbB-1 antibody complexed to the fusion protein. According toanother aspect, the invention provides a method for treating ErbB-1associated cancer in a subject, comprising the steps of administering tothe subject an effective amount of the composition comprising theanti-ErbB-1 antibody complexed to the fusion protein.

Embodiments of the fusion protein, the Fc-binding domain and thetruncated form of Pseudomonas exotoxin are as hereinbefore described.

According to a currently preferred embodiment, the fusion protein hasthe amino acid sequence of SEQ ID NO: 1. According to one embodiment,the antibody is a chimeric anti-ErbB-1 antibody. According to oneembodiment, the antibody is an IgG1. In one embodiment, the antibody isselected form the group consisting of a monoclonal antibody, a humanizedantibody, a chimeric antibody, a single chain antibody, and a fragmentthereof. According to a specific embodiment, the antigen recognized onthe target cells is selected from the group consisting of MUC-1 andErbB2. According to one embodiment, the subject is a mammal. Accordingto one embodiment, the mammal is a human.

In one embodiment, the target cell is a cancer cell. In one embodiment,the cancer cell is from a site selected from the group consisting oflung, colon, rectum, breast, ovary, prostate gland, head, neck, bone,kidney, liver, skin and the immune system. In one embodiment, the cancercell is a breast cancer cell.

According to one embodiment, the exposing in (d) is carried out in vivoor ex vivo. According to one embodiment, the exposing in (d) comprisesadministering the immunotoxin complex by a route selected from the groupconsisting of intravenous, intraperitoneal, subcutaneous, intramuscularand intralymphatic. According to one embodiment, step (c) comprisesforming covalent bonds between the antibody and the fusion protein. Inone embodiment, the step of forming covalent bonds comprises chemicalconjugation or cross-linking.

According to yet another aspect, the invention provides a method forassessing whether an antibody specific for a cell surface antigen isinternalized into target cells expressing the antigen, the methodcomprising:

-   -   a. providing a recombinant fusion protein, wherein the fusion        protein comprises a first segment which is the ZZ Fc-binding        domain derived from S. aureus protein A and a second segment        which is PE38 derived from Pseudomonas exotoxin;    -   b. selecting at least one antibody specific for the cell surface        antigen present on the target cells;    -   c. combining the antibody from (b) with the ZZ-PE38 fusion        protein from (a) to form a complex; and    -   d. exposing said target cells to the complex of (c); and    -   e. assessing whether the complex is internalized into the target        cells.        According to one embodiment, the fusion protein has the amino        acid sequence of SEQ ID NO: 1. According to another embodiment,        the fusion protein further comprises a detectable moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. is a schematic representation of mammalian pMAZ-IgH and pMAZ-IgLexpression vectors. Represented are maps of plasmids pMAZ-IgH for humanγ1 heavy chain expression and pMAZ-IgL for human κ light chainexpression carrying desired V genes for the production of human IgG1antibodies in mammalian cell culture.

FIGS. 2A to 2D are an analysis of stable clones expressing chimeric H23IgG and evaluation of antigen binding, as follows:

FIG. 2A shows dot-blot analysis of stable clones expressing chimericIgG. Supernatants (100 μl) of individual clones were applied induplicate onto a nitrocellulose filter. ChH23 IgG antibodies weredetected using HRP-conjugated goat anti-human IgG as secondary antibody.The membrane was developed using ECL reagents and exposure to X-rayfilm.

FIG. 2B is an immunoblot of spent culture medium of positive clones fordetermination of heavy and light chain production. Lanes 1-4, candidateclones; lane 5, commercial human IgG (0.5 μg/ml).

FIG. 2C is a graph showing an analysis of MUC1 binding by individualclones by ELISA. Microtiter plates were coated with MUC1-containingspent culture medium (100 μl/well). HRP-conjugated goat anti-human IgGwas used as secondary antibody. The ELISA was developed using thechromogenic HRP substrate TMB. The error bars represent standarddeviations of three independent experiments.

FIG. 2D is a blot analysis showing the determination of chH23 secretionlevels. Supernatants (100 μl) of stable clone B2 were applied in atwo-fold dilution series onto a nitrocellulose filter alongside atwo-fold dilution series of commercial human IgG standard at theindicated concentrations. HRP-conjugated goat anti-human IgG was used assecondary antibody.

FIGS. 3A to 3C are blot analyses showing the purification of chH23 andchFRP5 IgG1 from culture media of HEK293 cells, as follows:

FIG. 3A shows the purification of soluble chH23 IgG1 using protein Acolumn chromatography. Lane1, total culture media cell extract; lane 2,protein A column flow-through; lane 3, purified chH23 IgG1 clone B2antibody. Proteins were separated on a 12%/SDS polyacrylamide gel underreducing conditions and visualized by staining with GelCode-Blue®.Arrows mark the size and position of human IgG1 heavy and light chains.

FIG. 3B shows a Western-Blot analysis of purified chH23 IgG1 clone B2antibody using HRP-conjugated goat anti-human IgG.

FIG. 3C shows the purification of soluble chFRP5 IgG1 using protein Acolumn chromatography. Lane1, total culture media cell extract; lane 2,protein A column flow-through; lane 3, purified chFRP5 IgG1 clone G1antibody. Proteins were separated on a 12%/SDS polyacrylamide gel underreducing conditions and visualized by staining with GelCode-Blue®.

FIG. 4 is a graph showing an evaluation of MUC1 binding-affinity by H23Mab and chH23 IgG1 in ELISA. Comparative analysis of MUC1binding-affinity by chH23 IgG1 (filled squares) and murine H23 mAb(filled triangles) by half-maximal binding assay using standard ELISA.Microtiter plates were coated with MUC1-containing spent culture mediumin 50 mM NaHCO₃ (pH 9.6) buffer in a volume of 100 μl. MUC1 bindingassays were performed with 2 μg/ml of purified antibodies in two-folddilution series. HRP-labeled goat anti-human and HRP-labeled goatanti-mouse were used as secondary antibodies. The ELISA was developedusing the chromogenic HRP substrate TMB. The error bars representstandard deviations of three independent experiments. Thebinding-affinity was estimated as the IgG concentration that generates50% of the maximal signal.

FIGS. 5A and 5B are graphs showing an evaluation of MUC1 and ErbB2binding-affinities by whole-cell ELISA.

FIG. 5A is a comparative cellular MUC1 binding-affinity by chH23 IgG1(square symbols) and murine H23 mAb (triangle symbols) was assessed bywhole-cell ELISA. The human breast carcinoma T47D cell line was used asMUC1 expressing cells. To confirm specificity, antibodies (1 μg/ml) wereincubated in the presence (empty symbols) or absence (filled symbols) ofMUC1 protein prior to incubation with the cells.

FIG. 5B is a comparative cellular ErbB2 binding-affinity by chFRP5 IgG1(filled triangles) and Herceptin® mAb (filled squares) was assessed bywhole-cell ELISA. The human breast adenocarcinoma SKBR3 cell line wasused as ErbB2 expressing cells. Cells were incubated with antibodies (20μg/ml) for 1.5 h at 4° C. HRP-conjugated goat anti-human and HRP-labeledgoat anti-mouse antibodies were used as secondary antibodies. The ELISAwas developed using the chromogenic HRP substrate TMB. The error barsrepresent standard deviations of three independent experiments. Thebinding-affinity was estimated as the IgG concentration that generates50% of the maximal signal.

FIG. 6A (panels a and b) and 6B (panels a and b) are graphs showingflow-cytometry analyses of antibody binding to cells expressing MUC1.

FIG. 6A shows H23 mAb (panel a) and chH23 mAb (panel b) assessed forbinding to cellular MUC1 using the cell lines DA3 (murine), DA3-MUC1(MUC1-transfected DA3), T47D (human breast carcinoma), MCF7 (humanbreast carcinoma) and HEK293 (human kidney). To confirm specificity,antibodies (10 μg/ml) were incubated in the presence or absence of anexcess of MUC1 protein prior to incubation with the cells. Filled areas,negative control; bold black line, specific binding of antibody; blackline, competition for cell binding with soluble MUC1 protein.

FIG. 6B shows chFRP5 IgG1 (panel a) and Herceptin® mAb (panel b)assessed for binding to cellular ErbB2 using the cell lines SKBR3 (humanbreast adenocarcinoma), the A431 (human epidermoid carcinoma), T47D(human breast carcinoma), MCF7 (human breast carcinoma) and MDA-MB231(human mammary carcinoma). Cells were incubated with antibodies (10μg/ml) for 1.5 h at 4° C. FITC-conjugated goat anti-human andFITC-labeled goat anti-mouse antibodies were used as secondaryantibodies. Filled areas, negative control; black line, specific bindingof antibody.

FIGS. 7A to 7D are photomicrographs showing an analysis of antibodyinternalization confocal microscopy. Internalization of chH23 IgG1 intohuman breast carcinoma T47D cells and of chFRP5 IgG1 into SKBR3 cellsline was evaluated at 4° C. (FIGS. 7A and 7C, respectively) and at 37°C. (FIGS. 7 b and 7D, respectively) using confocal microscopy. Forevaluation at 4° C., cells were preincubated with complete mediumsupplemented with 0.5% NaN₃ for 2 h at 4° C., followed by the additionof antibody (5 μg/ml) and incubation for 1 h at 4° C. For evaluation at37° C., cells were incubated with antibody (5 μg/ml) in complete mediumfor 1 h at 37° C. in a humidified atmosphere of 95% air and 5% CO₂.

FIGS. 8A to 8C are blot analyses that show the purification of solubleZZ-PE38 fusion protein and immunoconjugates comprising ZZ-PE38, wherein:

FIG. 8A shows the purification of soluble ZZ-PE38. Purified ZZ-PE38fusion protein was obtained by subjecting periplasmic fractions to twosequential chromatography steps of Q-SEPHAROSE and MONO-Q anion exchangecolumns using fast protein liquid chromatography (FPLC). MW, molecularmass marker; lane 1, soluble ZZ-PE38 periplasm extract (15 μg); lane 2,purified ZZ-PE38 (5 μg).

FIG. 8B shows the purification of chH23-ZZ-PE38. ImmunoconjugatechH23-ZZ-PE38 was purified from a mixed sample containing excess ZZ-PE38and unbound chH23 by gel filtration using FPLC. MW, molecular massmarker; lane 1, purified chH23-ZZ-PE38 (2 μg).

FIG. 8C shows the purification of chFRP5-ZZ-PE38. ImmunoconjugatechFRP5-ZZ-PE38 was purified from a mixed sample containing excessZZ-PE38 and unbound chFRP5 gel filtration using FPLC. MW, molecular massmarker; lane 1, purified chFRP5-ZZ-PE38 (5 μg). In A, B and C, proteinswere separated on a 12%/SDS polyacrylamide gel under reducing conditionsand visualized by staining with GELCODE BLUE® dye. In B and C, thepositions of ZZ-P38 and antibody heavy and light chains are marked.

FIGS. 9A to 9C are analyses of chH23-ZZ-PE38, as follows:

FIG. 9A is a graph showing an analysis of MUC1 binding by chH23-ZZ-PE38in whole-cell ELISA. Human breast carcinoma T47D cells were incubatedwith 1 μg/ml of chH23-ZZ-PE38 or chH23 for 1.5 h at 4° C., washed andincubated with rabbit anti-PE sera mixed with either HRP-conjugated goatanti-rabbit antibody or with HRP-labeled goat anti-human antibody forthe detection of bound chH23-ZZ-PE38 immunotoxin (open triangles), orbound chH23 (filled triangles), respectively. The ELISA was developedusing the chromogenic HRP substrate TMB. The error bars representstandard deviations of three independent experiments.

FIGS. 9B and 9C are graphs showing flow-cytometry analyses ofchH23-ZZ-PE38 immunoconjugate. Cellular MUC1 binding activity wasevaluated on human breast carcinoma T47D cell line. Cells were incubatedwith 10 μg/ml of chH23-ZZ-PE38 (B) or control hIgG-ZZ-PE38 (C) for 1.5 hat 4° C. In (B), specific chH23-ZZ-PE38 binding was confirmed byincubation of the immunoconjugate in the presence or absence of 10-foldexcess of un-conjugated chH23 IgG1 prior to incubation with the cells.Rabbit anti-PE sera mixed with FITC-labeled goat anti-rabbit was usedfor the detection of bound chH23-ZZ-PE38 immunotoxin. Filled areas,negative control; black line, specific binding of antibody; grey line,competition for cell binding with chH23 IgG1.

FIGS. 10A and 10B are graphs showing the inhibition of the growth ofhuman tumor cell lines by chH23-ZZ-PE38 immunoconjugate. T47D (FIG. 10A)and MCF7 (FIG. 10B) tumor cells were incubated for 48 h with theindicated concentration of chH23-ZZ-PE38 (filled circles), hIgG-ZZ-PE38(open triangles) or ZZ-PE38 (filled squares). The relative number ofviable cells was determined using an enzymatic MTT assay and isindicated as the absorption at 570 nm. Each point represents the mean ofa set of data determined in triplicate in three independent experiments.The results are expressed as percentage of living cells respect to theuntreated controls that were processed simultaneously using thefollowing equation: (A₅₇₀ of treated sample/A₅₇₀ of untreatedsample)×100. The IC₅₀ values were defined as the immunotoxinconcentrations inhibiting cell growth by 50%.

FIG. 11 is a graph that illustrates an evaluation of ErbB2binding-affinity by whole-cell ELISA. Comparative cellular ErbB2binding-affinity by chFRP5-ZZ-PE38 (filled triangles) and scFv(FRP5)-ETA(filled diamonds) was tested by whole-cell ELISA. ErbB2 expressing humanbreast adenocarcinoma SKBR3 cells were incubated with 100 nm ofchFRP5-ZZ-PE38 or 1000 nm of scFv(FRP5)-ETA for 1.5 h at 4° C. Followingwashing steps, Rabbit anti-PE sera mixed with HRP-conjugated goatanti-rabbit were used for the detection of bound immunotoxins. The ELISAwas developed using the chromogenic HRP substrate TMB. The error barsrepresent standard deviations of three independent experiments. Thebinding-affinities were estimated as the immunotoxin concentration thatgenerates 50% of the maximal signal.

FIG. 12 (panels 1-5) are graphs showing flow cytometry analyses thatillustrate the comparison between chFRP5-ZZ-P38 and scFv(FRP5)-ETA forbinding to cellular ErbB2 using the cell lines SKBR3 (human breastadenocarcinoma; panel 1), A431 (human epidermoid carcinoma; panel 2),MCF7 human breast carcinoma; panel 3), T47D (human breast carcinoma;panel 4), and MDA-MB231 (human mammary carcinoma; panel 5). Cells wereincubated with 5 μm of each immunotoxin for 1.5 h at 4° C. Rabbitanti-PE sera mixed with FITC-labeled goat anti-rabbit were used for thedetection of bound immunotoxin. In each of panels 1-5: (A), filledareas, negative control; black line, specific binding of chFRP5-ZZ-P38;(B), filled areas, negative control; black line, specific binding ofscFv(FRP5)-ETA; (C), overlapping staining intensities of bothimmunotoxins, filled areas, negative control; bold black line, specificbinding of chFRP5-ZZ-P38; black line, specific binding ofscFv(FRP5)-ETA.

FIGS. 13A and 13B are graphs showing the flow cytometry analysis ofchH23-ZZ-PE38 immunoconjugate. Cellular ErbB2 binding specificity wasevaluated on human breast adenocarcinoma SKBR3 cell line. Cells wereincubated with 10 μg/ml of chFRP5-ZZ-PE38 (FIG. 13A) or controlhIgG-ZZ-PE38 (FIG. 13B) for 1.5 h at 4° C. In (FIG. 13B), specificchFRP5-ZZ-PE38 binding was confirmed by incubation of theimmunoconjugate in the presence or absence of 10-fold excess ofun-conjugated chFRP5 IgG1 prior to incubation with the cells. Rabbitanti-PE sera mixed with FITC-labeled goat anti-rabbit were used for thedetection of bound chFRP5-ZZ-PE38. Filled areas, negative control; blackline, binding of specific antibody; grey line, competition for cellbinding with chH23 IgG1.

FIGS. 14A through 14E are graphs showing the inhibition of the growth ofhuman tumor cell lines by the chFRP5-ZZ-PE38. SKBR3 (FIG. 14A), A431(FIG. 14B), T47D (FIG. 14C), MCF7 (FIG. 14D) and MDA-MB231 (FIG. 14E)tumor cells were incubated for 48 h with the indicated concentrations ofchFRP5-ZZ-PE38 (filled circles), hIgG-ZZ-PE38 (filled squares), chFRP5IgG1 (open circles) or ZZ-PE38 (open triangles). The relative number ofviable cells was determined using an enzymatic MTT assay and isindicated as the absorption at 570 nm. Each point represents the mean ofa set of data determined in triplicate in three independent experiments.The results are expressed as percentage of living cells in comparison tothe untreated controls that were processed simultaneously using thefollowing equation: (A₅₇₀ of treated sample/A₅₇₀ of untreatedsample)×100. The IC₅₀ values were defined as the immunoconjugateconcentrations inhibiting cell growth by 50%. The error bars representstandard deviation of three independent experiments.

FIGS. 15A through 15D are graphs showing comparative cytotoxicactivities of chFRP5-ZZ-PE38 and scFv(FRP5)-ETA. SKBR3 (FIG. 15A), A431(FIG. 15B), MCF7 (FIG. 15C) and MDA-MB231 (FIG. 15D) cells wereincubated for 48 h with the indicated concentrations of chFRP5-ZZ-PE38or scFv(FRP5)-ETA. To confirm specificity, both immunotoxins wereincubated in medium assay with 100 μg/ml of chFRP5 IgG1. The relativenumber of viable cells was determined using an enzymatic MTT assay andis indicated as the absorption at 570 nm. Filled circles,chFRP5-ZZ-PE38; filled squares, chFRP5-ZZ-PE38 incubated in the presenceof 100 μg/ml competing chFRP5 IgG1; open circles, scFv(FRP5)-ETA; opentriangles, scFv(FRP5)-ETA incubated in the presence of 100 μg/mlcompeting chFRP5 IgG1. Each point represents the mean of a set of datadetermined in triplicate in three independent experiments. The errorbars represent standard deviation of three independent experiments.

FIGS. 16A and 16B are graphs showing the effect of competing IgGs oncell-killing activity of chFRP5-ZZ-PE38. SKBR3 (FIG. 16A) and A431 (FIG.16B) cells were incubated for 48 h with the indicated concentrations ofchH23-ZZ-PE38 in the presence or absence of 100 μg/ml of Herceptin® mAbor chFRP5 IgG1 in assay medium. The relative number of viable cells wasdetermined using an enzymatic MTT assay and is indicated as theabsorption at 570 nm. Filled circles, chFRP5-ZZ-PE38; filled squares,chFRP5-ZZ-PE38 incubated in the presence of 100 μg/ml competingHerceptin® mAb; open circles, chFRP5-ZZ-PE38 incubated in the presenceof 100 μg/ml competing chFRP5 IgG1. Each point represents the mean of aset of data determined in triplicate in three independent experiments.The error bars represent standard deviation of three independentexperiments.

FIGS. 17A and 17B are graphs showing the analysis of ErbB2 binding bychH23-ZZ-PE38 following preincubation with competitors for ZZ binding.Cellular ErbB2 binding was evaluated by whole-cell ELISA on human breastadenocarcinoma SKBR3 cell line followed preincubation of chFRP5-ZZ-PE38at 37° C. in PBS with (FIG. 17A) 10-fold molar excess of protein-Apurified human IgG for periods up to 7 days, or (FIG. 17B) incubation in100% human serum of three individual donors. Rabbit anti-PE sera mixedwith HRP-conjugated goat anti-rabbit were used for the detection ofbound chFRP5-ZZ-PE38, while HRP-labeled goat anti-human was used for thedetection of bound chFRP5. The ELISA was developed using the chromogenicHRP substrate TMB. In A: Open squares, chFRP5; filled triangles,chFRP5-ZZ-PE38; open circles, chFRP5-ZZ-PE38 incubated in the presenceof x10 molar excess of competing human IgG. In (B): open squares, opentriangles and open circles represent chFRP5-ZZ-PE38 incubated in thepresence of ×10 molar excess of competing human IgG from three differentsources respectively. The error bars represent standard deviations ofthree independent experiments.

FIGS. 18A and 18B are analyses of crosslinked chFRP5-ZZ-PE38, wherein:

FIG. 18A is a blot analysis of crosslinked chFRP5-ZZ-PE38 by SDS/PAGE.MW, molecular mass marker, lane 1, purified, untreated chFRP5-ZZ-PE38immunoconjugate (3 μg); lane 2, unpurified crosslinked chFRP5-ZZ-PE38immunoconjugate (3 μg). Proteins were separated on a 10%/SDSpolyacrylamide gel under reducing conditions and visualized by stainingwith GelCode Blue®. Arrows mark the position of ZZ-P38 and chFRP5 heavyand light chains.

FIG. 18B is a graph showing the analysis of ErbB2 binding bycrosslinked-chH23-ZZ-PE38 in whole-cell ELISA. Comparative cellularErbB2 binding was evaluated by whole-cell ELISA on human breastadenocarcinoma SKBR3 cells following preincubation ofcrosslinked-chFRP5-ZZ-PE38 and chFRP5-ZZ-PE at 37° C. for 24 h in thepresence or absence of 10-fold molar excess of protein-A purified humanIgG antibodies or alternatively, in 100% human serum. Rabbit anti-PEsera mixed with HRP-conjugated goat anti-rabbit were used for thedetection of bound immunotoxins. Filled squares, chFRP5-ZZ-PE38; filledtriangles, chFRP5-ZZ-PE38 following incubation with competing humanserum; filled circles, chFRP5-ZZ-PE38 following incubation withcompeting human IgG; open squares, crosslinked chFRP5-ZZ-PE38; opentriangles, crosslinked chFRP5-ZZ-PE38 following incubation withcompeting human serum; open circles, crosslinked chFRP5-ZZ-PE38following incubation with competing human IgG. The ELISA was developedusing the chromogenic HRP substrate TMB. The error bars representstandard deviations of three independent experiments.

FIGS. 19A and 19B are graphs showing the cell killing activity ofcrosslinked-chH23-ZZ-PE38 following preincubation with human IgG orhuman serum. Crosslinked-chH23-ZZ-PE38 and chH23-ZZ-PE38 werepreincubated at 37° C. for 24 h in the presence or absence of (FIG. 19A)10-fold molar excess of protein-A purified human IgG antibodies, or(FIG. 19B) incubated for 24 h in 100% human serum before being evaluatedfor their cytotoxic activity on A431 cells. The relative number ofviable cells was determined using an enzymatic MTT assay and isindicated as the absorption at 570 nm. Filled circles, chFRP5-ZZ-PE38;filled squares, chFRP5-ZZ-PE38 following incubation with competitor;open circles, crosslinked chFRP5-ZZ-PE38; open squares, crosslinkedchFRP5-ZZ-PE38 following incubation with competitor. Each pointrepresents the mean of a set of data determined in triplicate in threeindependent experiments. The error bars represent standard deviations ofthree independent experiments.

FIGS. 20A and 20B are blot analyses showing a determination ofchFRP5-ZZ-PE38 and scFv(FRP5)-ETA serum concentration by dot-blotanalysis. 100 μl of serum samples diluted 1:100 in PBS of mice injectedwith chFRP5-ZZ-PE38 (FIG. 20A) or scFv(FRP5)-ETA (FIG. 20B) were appliedin a two-fold dilution series as dots onto a nitrocellulose filteralongside a two-fold dilution series of 100 ng/ml of chFRP5-ZZ-PE38(FIG. 20A) and scFv(FRP5)-ETA (FIG. 20B) that were used to determine theimmunotoxin concentration in each serum sample. Rabbit anti-PE mixedwith HRP-conjugated goat anti-rabbit were used for the detection of theimmunotoxins. The membrane was developed using ECL reagents and exposureto X-ray film.

FIG. 21 is a graph that shows blood levels of chFRP5-ZZ-PE38 andscFv(FRP5)-ETA in mice. Female BALB/c mice were given a single molarequivalent i.v. dose of 15 μg chFRP5-ZZ-PE38 or 5 μg scFv(FRP5)-ETA byinjection into the tail vein. Blood samples were drawn from the orbitalvein of mice injected with scFv(FRP5)-ETA at 2, 5, 10, 20, 30, 60, 120and 240 min after injection and at 2, 5, 10, 20, 30, 60, 120, 240, 480and 1440 min for mice injected with chFRP5-ZZ-PE38. The immunotoxinsblood levels at each time point was calculated by dot-blot analysis ofserum dilutions and by measuring cell-killing activity of serumdilutions on A431 cells using MTT assay. Results are the mean from twomice for each time point±SE. Filled circles, chFRP5-ZZ-PE38; filledtriangles, scFv(FRP5)-ETA. Insert: expanded view of results from thefirst 4 hrs.

FIG. 22 is a graph that shows the antitumor effect of chFRP5-ZZ-PE38 andscFv(FRP5)-ETA on the in vivo growth of A431 tumor xenografts in nudemice. Groups of 3-5 mice were given s.c. injections of 1.5×10⁶ A431cells on day 0, and were treated by i.v. injections of 0.5 mg/kgchFRP5-ZZP-E38 (open squares) or 0.25 mg/kg chFRP5-ZZ-PE38 (filledtriangles) or scFv(FRP5)-ETA (open triangles) on days 9,12,15,18 and 21marked by arrows when tumors were established. Control mice were treatedwith PBS (filled squares). Tumor size was measured at the indicated timepoints and tumor volumes were calculated. The mean values for each groupare shown and the standard deviation is represented by error bars.

FIG. 23 are photographs illustrating xenograft-bearing nude micephotographed on day 30 of the anti-tumor experiment. Shown are 2 mice ofeach group: A, control (PBS injected) mice. B, scFv(FRP5)-ETA (0.25mg/kg) injected mice. C, chFRP5-ZZ-PE38 (0.25 mg/kg) injected mice. D,chFRP5-ZZ-PE38 (0.5 mg/kg) injected mice. Arrows mark location oftumors.

FIG. 24 is a micrograph of a Poly Acrylamide Gel Electrophoresis SDSPAGE showing bands of non-cross-linked and cross-linked cetuximab-ZZPE38 complex. Lane 1 contains ZZ-PE38 in periplasmic fraction beforeenrichment; Lane 2 contains ZZ-PE38 in periplasmic fraction afterenrichment; lane 3 contains cetuximab; lane 4 contains cetuximab-ZZ-PE38complex (non-cross linked); and lane 5 contains cetuximab-ZZ-PE38complex (cross linked).

FIG. 25 contains graphs showing the binding efficiency of cetuximab tohead and neck (H & N) cancer cells (SCC-1, SCC-9 and KB) and prostatecancer cells (C1-1-3 and C1-1-7). FIG. 25A shows FACS analysis graph ofnon-stained SCC-1 cells and Cetuximab-stained SCC-1 cells. FIG. 25Bshows an ELISA graph of the antibody binding assay in in SCC-1 and SCC-9cells. FIG. 25C shows FACS analysis graph of non-stained CI-1 cells andCetuximab-stained CI-1 cells. FIG. 25D shows an ELISA graph of theantibody binding assay in CI-1-3 and CI-1-7 cells.

FIG. 26 is a graph showing the survival rate of SCC-1, ErbB-1 expressingcell exposed to cetuximab-ZZ-PE38 complex, human IgG-ZZ-PE38 complex, orZZ-PE38 alone.

FIG. 27 contains graphs demonstrating the cell survival inhibitoryeffect of cetuximab-ZPE38 complex on H&N(SCC-1 and SCC-9, FIG. 27A) andprostate (C1-1-3 and C1-1-7, FIG. 27B) cancer cells with differentErbB-1 expression (shown by Western blot analysis micrographs in FIGS.27C and 27D, respectively) as well as on survival of normal fibroblastswith very low ErbB-1 expression (demonstrated by ELISA in FIGS. 27E and27F, respectively).

FIG. 28 contains graphs demonstrating the SCC-1 cell survival inhibitoryeffect of cetuximab-ZZ-PE38 complex in the presence and absence of humanIgG and compared to IgG-ZZ-PE38 complex.

FIG. 29 contains graphs comparing the tumor size inhibitory effect ofcetuximab, ZZ-PE38, and cetuximab-ZZ-PE38 complex.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a polynucleotide construct encoding afusion protein, said polynucleotide construct comprising a firstsequence encoding an immunoglobulin Fc-binding domain, for example thesynthetic Fc-binding domain derived from Staphylococcus aureus protein Adenoted as “ZZ”, and a second sequence encoding a truncated form ofPseudomonas exotoxin (PE), and fusion proteins encoded by saidconstruct. Moreover, the present invention relates to: (1) a fusionprotein comprising a first sequence encoding an immunoglobulinFc-binding domain, for example the synthetic Fc-binding domain derivedfrom Staphylococcus aureus protein A denoted as “ZZ”, and a secondsequence encoding a truncated form of Pseudomonas exotoxin (PE); and (2)an anti-ErbB-1 antibody—an immunotoxin.

The invention also relates to the preparation of immunotoxin conjugatesusing the recombinant fusion protein ZZ-PE and efficiently internalizingIgGs, IgG complexes and Fc-fusion proteins. Immunocomplexesincorporating ZZ-PE and efficiently internalizing IgG are highlyspecific and potent immunotoxins. Also encompassed is the preparation ofimmunotoxin conjugates using the recombinant fusion protein ZZ-PE andefficiently internalizing anti-ErbB-1 IgGs, IgG complexes and Fc-fusionproteins. Immunocomplexes incorporating ZZ-PE and efficientlyinternalizing anti-ErbB-1 IgG are highly specific and potentimmunotoxins.

DEFINITIONS

As used herein, an “immunoglobulin Fc-binding domain” refers to aprotein or peptide which binds the Fc region of an immunoglobulinmolecule. For example, S. aureus protein A (SPA) and various fragmentsthereof bind a specific region on the Fc of human IgG1 and other Igmolecules.

The terms “polynucleotide construct” and “polynucleotide sequence” areused herein interchangeably to refer to a polymer of nucleotides, suchas deoxyribonucleotides, ribonucleotides, or modified forms thereof inthe form of an individual fragment or as a component of a largerconstruct, in a single strand or in a double strand form. Thepolynucleotides to be used in the invention include sense and antisensepolynucleotide sequences of DNA or RNA as appropriate to the goals ofthe therapy practiced according to the invention. The DNA or RNAmolecules may be complementary DNA (cDNA), genomic DNA, synthesized DNAor a hybrid thereof or an RNA molecule such as mRNA. Accordingly, asused herein, the terms “DNA construct”, “gene construct” and“polynucleotide” are meant to refer to both DNA and RNA molecules.

The terms “recombinant fusion protein” and “fusion protein” are usedherein interchangeably to refer to a protein produced by recombinanttechnology which comprises segments i.e. amino acid sequences, fromheterologous sources, such as different proteins or different organisms.The segments are joined either directly or indirectly to each other viapeptide bonds. By indirect joining it is meant that an intervening aminoacid sequence, such as a peptide linker is juxtaposed between segmentsforming the fusion protein.

Accordingly, the fusion protein of the invention may optionally comprisea peptide linker. The fusion protein may contain two or more segments.In the case of a fusion protein having more than two segments, someadjacent segments may be directly joined without any peptide linker,while other adjacent segments may be joined via a peptide linker. Asused herein, “recombinant fusion protein” and “recombinant protein”include reference to a protein produced using cells that do not have, intheir native state, an endogenous copy of the DNA able to express theprotein. The cells produce the recombinant protein because they havebeen genetically altered by the introduction of the appropriate isolatednucleic acid sequence. In accordance with the invention, a recombinantfusion protein is encoded by the polynucleotide construct as disclosedherein.

The term also includes reference to a cell, or nucleic acid, or vector,that has been modified by the introduction of a heterologous nucleicacid or the alteration of a native nucleic acid to a form not native tothat cell, or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found withinthe native (non-recombinant) form of the cell, express mutants of genesthat are found within the native form, or express native genes that areotherwise abnormally expressed, underexpressed or not expressed at all.

The term “antibody” as used herein refers to immunoglobulin (Ig)molecules and immunologically active portions of Ig molecules, i.e.molecules that contain an antigen binding site that specifically binds(immunoreacts with) an antigen. Such antibodies include, but are notlimited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab′and F(ab′)₂ fragments, and an Fab expression library. In general,antibody molecules obtained from humans relates to any of the classesIgG, IgM, IgA, IgE and IgD, which differ from one another by the natureof the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG1, IgG2, and others. Furthermore, inhumans the light chain may be a kappa chain or a lambda chain. Referenceherein to antibodies includes a reference to all such classes,subclasses and types of human antibody species.

The terms “selectively reactive”, “specific binding”, “specificrecognition” and related grammatical forms thereof are used hereininterchangeably to refer to the preferential association of an antibody,in whole or part, with a cell or tissue bearing a particular antigen andnot to cells or tissues lacking that antigen. It is, of course,recognized that a certain degree of non-specific interaction may occurbetween an antibody and a non-target cell or tissue. Nevertheless,selective reactivity may be distinguished as mediated through specificrecognition of the antigen. Although selectively reactive antibodiesbind antigen, they may do so with low affinity. On the other hand,specific binding results in a much stronger association between theantibody and cells bearing the antigen than between the bound antibodyand cells lacking the antigen. Specific binding typically results ingreater than 2-fold, preferably greater than 5-fold, more preferablygreater than 10-fold and most preferably greater than 100-fold increasein amount of bound antibody (per unit time) to a cell or tissue bearingthe antigen recognized by the preferred antibody as compared to a cellor tissue lacking expression of the antigen. Specific binding to aprotein under such conditions requires an antibody that is selected forits specificity for a particular protein. A variety of immunoassayformats are appropriate for selecting antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow & Lane,Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork (1988), for a description of immunoassay formats and conditionsthat can be used to determine specific immunoreactivity.

Staphylococcus aureus protein A (also referred to herein as “protein A”,“Staphlococcal protein A” and “SPA”) in its native form is a 42 kDpolypeptide which exhibits strong binding to the Fc region of many IgGmolecules, including human IgG1, IgG2 and IgG4 and mouse IgG2a andIgG2b, without interfering with the antigen binding site (Surolia et alTrends Biochem. Sci. 7 (1981):74; Lindmark et al, J. Immunol. Meth. 62(1983):1).

The fusion protein according to the invention comprises animmunoglobulin Fc-binding domain. A suitable immunoglobulin Fc-bindingdomain is one which is derived from S. aureus protein A, for example thetandem repeated, mutated form of domain B termed ZZ (SEQ ID NO:2).Disclosures of derivatives of protein A are provided for example inNilsson et al 1987, Protein Eng. 1, 107-13; Nilsson et al 1996, ProteinEng. 1, 107-13, and Brasted and Wells (1996) Proc Natl Acad Sci USA 93,5688-92. The Fc-binding domain may be present in the fusion protein insingle or multiple copies. The immunoglobulin Fc-binding domain may bean anti-Fc single-chain antibody, as disclosed for example inAzriel-Rosenfeld et al 2004, J Mol Biol 335, 177-92.

Pseudomonas exotoxin (PE) in its native form is a monomeric protein of613 amino acids (molecular weight 66 kD) secreted by Pseudomonasaeruginosa, which inhibit protein synthesis in eukaryotic cells throughthe inactivation of elongation factor 2 (EF-2) by catalyzing itsADP-ribosylation i.e. the transfer of the ADP ribosyl moiety of oxidizedNAD onto EF-2.

The native PE sequence as disclosed for example in U.S. Pat. No.5,602,095, is provided herein as SEQ ID NO:4. The exotoxin containsthree structural domains that act in concert to cause cytotoxicity.Domain Ia (amino acids 1-252) mediates cell binding. Domain II (aminoacids 253-364) is responsible for translocation into the cytosol anddomain III (amino acids 400-613) mediates ADP ribosylation of elongationfactor 2. The function of domain Ib (amino acids 365-399) remainsundefined, although a large part of it, amino acids 365-380, can bedeleted without loss of cytotoxicity. See Siegall et al., J. Biol. Chem.264:14256-14261, 1989.

The term “Pseudomonas exotoxin” (“PE”) as used herein refers asappropriate to a full-length native (naturally occurring) PE or to a PEthat has been modified, for example a deletion mutant.

As used herein, the term “a truncated form of Pseudomonas exotoxin”refers to any mutant form of PE which comprises a deletion of a portionof the native sequence of PE and retains cytotoxic activity. Thus, thePE used in the invention disclosed herein includes fragments of thenative sequence, internal deletion mutants, conservatively modifiedvariants of native PE and fragments thereof, and combinations thereof,with the condition that such forms of PE are cytotoxic with or withoutsubsequent proteolytic or other processing in the target cell (e.g., asa protein or pre-protein). Such mutant forms may further compriseadditional sequences, modified amino acids and other variations as areknown in the art.

According to various embodiments, truncated forms of PE for use in theinvention include, without limitation PE38 (SEQ ID NO:3); PE38 KDEL (SEQID NO:23); PE38RDEL (SEQ ID NO:24); PE37 (SEQ ID NO:25) and PE40 (SEQ IDNO:26).

PE38 is composed of amino acids 253-364 and 381-613 of native PE whichis activated to its cytotoxic form upon processing within a cell (seee.g., U.S. Pat. No. 5,608,039, and Pastan et al 1997, Biochim. Biophys.Acta 1333:C1-C6). PE37 corresponds to amino acids 281-613 of native PElinked to an initial methionine residue, as disclosed for example inU.S. Pat. No. 5,602,095. PE38 KDEL corresponds to amino acids 253-364and 381-609 of native PE linked to the altered C-terminal sequence KDEL,as disclosed for example in Brinkmann et al 1991, Proc Nat Acad Sci USA88:8616-8621. PE38RDEL corresponds to amino acids 253-364 and 381-609 ofnative PE linked to the altered C-terminal sequence RDEL as disclosedfor example in Kreitman and Pastan 1995, Biochem J 307:29-37. PE40corresponds to amino acids 253-613 of native PE U.S. Pat. No. 6,051,405.

The invention also encompasses additional truncated forms of PE andvariants there of as are known in the art inter alia in the publicationscited supra.

The fusion protein of the invention comprises an immunoglobulinFc-binding domain, for example the synthetic ZZ domain derived from S.aureus protein A (SEQ ID NO:2), and a truncated form of Pseudomonasexotoxin such as PE38 (SEQ ID NO:3). In a currently preferredembodiment, the fusion protein consists of ZZ (SEQ ID NO:2) and PE38(SEQ ID NO:3) joined by a peptide linker (SEQ ID NO:27). The twosegments may be directly linked, or indirectly linked via an interveningsequence such as a peptide linker. It is generally preferable that anyintervening sequences are substantially devoid of any biologicalactivity. A suitable peptide linker is for example one having from aboutfour to about 20 amino acids. It may in some cases be preferable thatthe linker is composed substantially of neutral amino acids. A suitablelinker may for example have the sequence of SEQ ID NO:27. Many peptidelinkers are known in the art and may be used alternately oradditionally.

In a currently preferred embodiment, the fusion protein comprises orconsists ZZ (SEQ ID NO:2) and PE38 (SEQ ID NO:3) joined by a peptidelinker (SEQ ID NO:27). In a currently preferred embodiment, the fusionprotein is ZZ-PE38 and comprises the amino acid sequence of SEQ ID NO:1.In another embodiment, the present invention provides a DNA moleculeencoding the fusion protein comprising SEQ ID NO: 1. In anotherembodiment, the nucleotide sequence which encodes ZZ-PE38 is SEQ IDNO:6.

The term “monoclonal antibody” (mAb) as used herein, refers to apopulation of antibody molecules that contain only one molecular speciesof antibody molecule consisting of a unique light chain gene product anda unique heavy chain gene product. In particular, the complementaritydetermining regions (CDRs) of the monoclonal antibody are identical inall the molecules of the population. mAbs thus contain an antigenbinding site capable of immunoreacting with a particular epitope of theantigen characterized by a unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein, 1975, Nature, 256:495. In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes can beimmunized in vitro.

Additional types of antibodies suitable for use in the invention includehumanized antibodies and human or chimeric antibodies. These antibodiesare suitable for administration to humans without engendering an immuneresponse by the human against the administered immunoglobulin. Humanizedforms of antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab)₂ or otherantigen-binding subsequences of antibodies) that are principallycomprised of the sequence of a human immunoglobulin, and contain minimalsequence derived from a non-human immunoglobulin. Humanization can beperformed following the method of Winter and co-workers (Jones et al.,Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. (See also U.S. Pat. No. 5,225,539). Insome instances, Fv framework residues of the human immunoglobulin arereplaced by corresponding non-human residues. Humanized antibodies canalso comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the framework regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin (Presta, 1992, Curr. Op.Struct. Biol., 2:593-596).

Fully human antibodies essentially relate to antibody molecules in whichthe entire sequence of both the light chain and the heavy chain,including the CDRs, arise from human genes. Such antibodies are termed“human antibodies”, or “fully human antibodies” herein. Human monoclonalantibodies can be prepared by the trioma technique; the human B-cellhybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) andthe EBV hybridoma technique to produce human monoclonal antibodies (seeCole, et al., 1985 In: Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized inthe practice of the present invention and may be produced by using humanhybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B-cells with Epstein Barr Virus invitro (see Cole, et al., 1985 In: Monoclonal Antibodies And CancerTherapy, Alan R. Liss, Inc., pp. 77-96).

In addition, human antibodies can also be produced using additionaltechniques, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)). Similarly, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.(Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859(1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (NatureBiotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14,826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13:65-93(1995)).

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's endogenous antibodies in response to challengeby an antigen. (See PCT publication WO94/02602). The endogenous genesencoding the heavy and light immunoglobulin chains in the nonhuman hosthave been incapacitated, and active loci encoding human heavy and lightchain immunoglobulins are inserted into the host's genome. The humangenes are incorporated, for example, using yeast artificial chromosomescontaining the requisite human DNA segments. An animal which providesall the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. The preferred embodiment of such anonhuman animal is a mouse, and is termed the Xenomouse™, as disclosedin PCT publications WO 96/33735 and WO 96/34096. This animal produces Bcells which secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with an immunogenof interest, as for example, a preparation of a polyclonal antibody, oralternatively from immortalized B cells derived from the animal, such ashybridomas producing monoclonal antibodies. Additionally, the genesencoding the immunoglobulins with human variable regions can berecovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogs of antibodies such as, for example,single chain Fv molecules.

The term “expression cassette” refers to a recombinant nucleic acidconstruct comprising an expression control sequence operatively linkedto an expressible nucleotide sequence. An expression cassette generallycomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in vitro expressionsystem.

The invention further provides a recombinant expression vectorcomprising a polynucleotide construct of the invention cloned into theexpression vector. The term “expression vector” refers to a vectorcomprising an expression cassette. Expression vectors include all thoseknown in the art, such as cosmids, plasmids (e.g., naked or contained inliposomes) and viruses that incorporate the expression cassette. An“expression plasmid” comprises a plasmid nucleotide sequence capable ofdirecting a molecule of interest, which is operably linked to apromoter.

As used herein the term “operably linked” wherein referring to a firstnucleic acid sequence which is operably linked with a second nucleicacid sequence refers to a situation when the first nucleic acid sequenceis placed in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter effects the transcription or expression of thecoding sequence. Generally, operably linked nucleic acid sequences arecontiguous and, where necessary to join two protein coding regions, theopen reading frames are aligned.

A “promoter” is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see e.g., Bitter etal 1987, Methods in Enzymology 153:516-544). For example, when cloningin bacterial systems, inducible promoters such as pL of bacteriophagelambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may beused. In one embodiment, when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

By “host cell” is meant a cell into which (or into an ancestor of which)has been introduced, by means of recombinant DNA techniques, theexpression vector of the present invention. Transformation of a hostcell with recombinant DNA may be carried out by conventional techniquesas are well known to those skilled in the art. Where the host isprokaryotic, such as E. coli, cells which are competent for DNA uptakecan be prepared from cells harvested after exponential growth phase andsubsequently treated by using procedures well known in the art. Forexample, host cells can be made competent for transformation by CaCl₂,MgCl₂ or RbCl treatment. Alternatively, cells can be made competent forelectroporation by removing all traces of salt.

The invention further provides an immunotoxin conjugate (also referredto herein as an “immunoconjugate”), by which is meant a conjugate orcomplex comprising a targeting_moiety, typically based onantibody-antigen recognition, and a toxic moiety_which renders theimmunotoxin cytotoxic to the cells of interest.

The targeting moiety is any biological substance endowed with specificbinding properties towards a selected target cell. In one preferredembodiment, the targeting moiety is an ERbB-1 antibody-based moiety,including, but not limited to: monoclonal antibodies, polyclonalantibodies, chimeric antibodies, humanized antibodies and antibodyfragments such as recombinant antibody fragments, single-chainantibodies (scFv), single antibody variable domains, dsFv, Fab, F(ab′)₂,and the like as are known in the art. Single-chain antibodies are smallrecognition units consisting of the variable regions of theimmunoglobulin heavy (V_(H)) and light (V_(L)) chains which areconnected by a synthetic linker sequence. Single antibody domainproteins (dAbs) are minimized antibody fragments comprising either anindividual V_(L) domain or an individual V_(H) domain.

In another preferred embodiment, the targeting moiety is a peptideendowed with binding specificity towards the target cell (linear,circularly constrained or cyclic) or a short peptide selected from alibrary of short peptide sequences that is endowed with bindingspecificity towards the target cell. In another embodiment, thetargeting moiety is a peptide or an antibody which specificity binds anextracellular or an intracellular cancerous epitope. In anotherpreferred embodiment, the cancerous epitope is ErbB-1. In anotherembodiment, the targeting moiety is a monoclonal antibody whichspecifically binds an extracellular or an intracellular cancerousepitope. In another preferred embodiment, the cancerous epitope isErbB-1. In another preferred embodiment, the targeting moiety is amonoclonal antibody which specifically binds ErbB-1. In anotherpreferred embodiment, the targeting moiety is a chimeric monoclonalantibody which specifically binds ErbB-1. In another preferredembodiment, the targeting moiety is cetuximab. In another preferredembodiment, the targeting moiety is a chimeric (mouse/human) monoclonalantibody.

Methods for constructing libraries and using them for screening forligands having an affinity to a selected target molecule or cell areknown in the art. The targeting moiety may be a polypeptide, acarbohydrate, a lipid, a glycolipid, a saccharide, a nucleic acid andthe like, which is able to selectively bind a target molecule on atarget cell. For instance, the ligand may include known ligands of cellsurface receptors, or any natural or synthetic derivative thereof. Thetoxic moiety is the portion of an immunotoxin, which renders theimmunotoxin cytotoxic to cells of interest. The toxic moiety ispreferably derived from a toxin of plant or bacterial origin.

Specific, non-limiting examples of toxins include, but are not limitedto, abrin, ricin, Pseudomonas exotoxin (PE, such as PE37, PE38, andPE40), diphtheria toxin (DT), saporin, restrictocin, or modified toxinsthereof, or other toxic agents that directly or indirectly inhibit cellgrowth or kill cells. For example, PE and DT are highly toxic compoundsthat typically bring about death through liver toxicity. PE and DT,however, can be modified into a form for use as an immunotoxin byremoving the native targeting component of the toxin (e.g., domain Ia ofPE or the B chain of DT) and replacing it with a different targetingmoiety, such as an antibody.

According to the present invention, the toxic moiety is a component of afusion protein wherein the fusion protein comprises a first segmentwhich is an Fc-binding domain, and a second segment which is a truncatedform of Pseudomonas exotoxin, as described herein. According to acurrently preferred embodiment, the toxic moiety is the truncated formof Pseudomonas exotoxin denoted as PE38 which has the amino acidsequence of SEQ ID NO:3. In one embodiment, the toxic moiety PE38 is acomponent of the fusion protein ZZ-PE38 (SEQ ID NO:1).

The targeting moiety is preferably an antibody molecule, a chimericantibody molecule, fragment or other derivative or antibody complexwhich includes the Fc region of IgG, in particular the protein A bindingsite present thereon. Accordingly, the fusion protein of the inventionmay be complexed with the targeting moiety via interaction between theFc-binding domain of the former and the Fc region of the latter, to thusform an immunotoxin conjugate. Such a conjugate formed by combination ofthe two entities may be referred to as an immuno complex.

In cases where the targeting moiety does not include the Fc region ofIgG, for example in the case of an antibody fragment such as F(ab′)₂,the targeting moiety may be synthetically fused to or conjugated with anFc region, thereby conferring on the targeting moiety the ability toform a complex with the fusion protein.

The protein A mediated non-covalent interaction between the fusionprotein and the antibody may be sufficiently tight to enable use of thecomplex as an immunotoxin without further manipulation. In some caseshowever, it may be useful to introduce covalent bonds between the twoentities, for example by forming a chemically conjugated complex or across-linked complex.

“Covalent association”, “covalent bond” and associated grammaticalforms, such as “covalently associated” and “covalently bound”respectively, refer interchangeably to an intermolecular association orbond which involves the sharing of electrons in the bonding orbitals oftwo atoms. “Non-covalent association”, “non-covalent bond” andassociated grammatical forms refer interchangeably to intermolecularinteraction among two or more separate molecules or molecular entitieswhich does not involve a covalent bond. Intermolecular interaction isdependent upon a variety of factors, including, for example, thepolarity of the involved molecules, and the charge (positive ornegative), if any, of the involved molecules. Non-covalent associationsare selected from ionic interactions, dipole-dipole interactions, vander Waal's forces, and combinations thereof.

Methods for chemical conjugation and cross-linking are known in the art.A number of reagents capable of cross-linking molecules such as peptidesare known in the art, including for example, azidobenzoyl hydrazide,N-[4-(p-azidosalicylamino)butyl]-3′-[2′-pyridyldithio]propionamide),bis-sulfosuccinimidyl suberate, dimethyladipimidate,disuccinimidyltartrate, N-.gamma.-maleimidobutyryloxysuccinimide ester,N-hydroxy sulfosuccinimidyl-4-azidobenzoate,N-succinimidyl[4-azidophenyl]-1,3′-dithiopropionate,N-succinimidyl[4-iodoacetyl]aminobenzoate, glutaraldehyde, formaldehydeand succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate.

According to one embodiment, an immunotoxin comprises a complex of thefusion protein of the invention and an antibody, wherein the antibody iscapable of specifically binding an antigen expressed on the surface of atarget cell. In a preferred embodiment, the antibody is capable of beinginternalized into the target cell. In another preferred embodiment, theantibody is capable of specifically binding ErbB-1 on a target cell thusenabling the internalization of the immunotoxin into the target cell. Inanother preferred embodiment, the antibody is cetuximab (Erbitux®).

The target cell may be a cancer cell or a pathogen infected cell. Thepathogen may be a parasite or a virus. In another preferred embodiment,the target cell expresses ErbB-1. In another preferred embodiment, thetarget cell is a tumor cell expressing ErbB-1. In another preferredembodiment, the target cell is a tumor cell expressing ErbB-1 havingincreased resistance to chemo-radiotherapy. In another preferredembodiment, the target cell is a head and neck (H&N) cancer cell,colo-rectal cancer cell, prostate cancer cell, lung cancer cell or anyother cancer cell expressing ErbB-1 (epidermal growth factor receptor(EGFR).

In another embodiment, provided herein a synergistic targeted cancercomposition and a synergistic targeted cancer method of treatment, whichare based on the combination of an anti-ErbB-1 antibody and a fusionprotein such as described herein. In another embodiment, provided hereina synergistic targeted cancer composition and a synergistic targetedcancer method of treatment, which are based on the combination of ananti-ErbB-1 antibody and zz-PE38 (immunoconjugate). In anotherembodiment, provided herein a synergistic targeted cancer compositionand a synergistic targeted cancer method of treatment, which are basedon the combination of an anti-ErbB-1 monoclonal antibody and fusionprotein such as but not limited to zz-PE38 (immunoconjugate). In anotherembodiment, provided herein a synergistic targeted cancer compositionand a synergistic targeted cancer method of treatment, which are basedon the combination of cetuximab and zz-PE38. In another embodiment, itwas surprisingly found that the combination of cetuximab and zz-PE38 notonly inhibited tumor growth or decreased tumor growth rate but alsoinduced de-novo tumor shrinkage. In another embodiment, the presentinvention addresses a long-felt need in cancer therapy thus providing atargeted toxin in quantities that are safe to the surrounding healthytissues.

In another embodiment, the targeted cancer composition and the targetedcancer method of treatment described herein are used for eliminatingcancer cells, tumors, and/or metastasis that express ErbB-1. In anotherembodiment, the targeted cancer composition and the targeted cancermethod of treatment described herein are used for eliminating cancercells, tumors, and/or metastasis highly expressing ErbB-1. The presentinvention provides that anti-ErbB-1 antibodies such as cetuximab have alimited efficiency in treating ErbB-1 expressing cancers, however,combining anti-ErbB-1 antibodies with a fusion protein such as describedherein results in an unexpected inhibitory/cell eliminating synergisticeffect. In another embodiment, the present invention provides that theimmunotoxin is more effective in reducing tumor size, treating cancer,inhibiting metastasis, or inducing cancer cell death then the use ofeither a fusion protein (such as ZZ-PE38) alone or a fusion proteincombined with non specific IgG.

In another embodiment, the synergistic targeted cancer composition andthe synergistic targeted cancer method of treatment are apparent incancer cells/tumors that have higher level of ErbB-1.

The antigen targeted by the targeting antibody may be a tumor-associatedantigen, a pathogen-associated antigen, a parasite-associated antigen ora virus-associated antigen. Examples of tumor-associated antigensinclude without limitation MUC1 and ErbB2. The cancer cell may be thatof a cancer at a site selected from the group consisting of lung, colon,rectum, breast, ovary, prostate gland, head, neck, bone, kidney, liver,skin and the immune system. The cancer cell may be that of a cancer at asite selected from the group consisting of lung, colon, rectum, breast,ovary, prostate gland, head, neck, bone, kidney, liver, skin and theimmune system.

It is intended that the immunotoxin delivers a toxic moiety to thetarget ErbB-1 expressing cells, and not to cells which are healthy andor found in other sites of an organism. Accordingly, the desiredtoxicity is in relation to a specific tissue or cell set. In contrast,the terms “excessive toxicity”, “undesired toxicity” and the like referto toxicity against non-target cells.

The invention further provides a method for selectively killing anErbB-1 expressing target cell in a subject in need thereof, involvinguse of the recombinant fusion protein described herein.

Various methods, such as library screening, are known in the art forobtaining antibodies having specificity for antigens of interest.Various candidate antibodies may be obtained by such methods. Theability of one or more candidate antibodies to be internalized into atarget cell may then be assessed to determine if any particular antibodymay be used as the targeting component of a potential immunotoxin. Oncesuch an internalizing antibody has been selected, it may be combinedwith the fusion protein described herein so as to form an immunotoxincomplex. The immunotoxin complex may be formulated into a pharmaceuticalcomposition for administering to cells of the subject (either in vivo orex vivo), thereby selectively killing the target cell in the subject. Inanother embodiment a subject is a mammal. In another embodiment asubject is a rodent. In another preferred embodiment, the subject is ahuman.

The subject may be a mammal, in particular a human.

In one embodiment, the target cell is a cancer cell. In one embodiment,the cancer cell is from a site selected from the group consisting oflung, colon, rectum, breast, ovary, prostate gland, head, neck, bone,kidney, liver, skin and the immune system. In one embodiment, the cancercell is a breast cancer cell. In another embodiment, the target cell isa cancer cell expressing ErbB-1. In another embodiment, the target cellis a cancer cell over-expressing ErbB-1. In another embodiment, thetarget cell is a cancer cell characterized by ErbB-1 over-activity. Inanother embodiment, one of average skill in the art can readilydetermine over-activity or over-expression of ErbB-1 in a cell. Inanother embodiment, the target cell is an ErbB-1 associated cancer cell.In another embodiment, the target cell is a cell over-expressing ErbB-1.In one embodiment, the cancer cell is a breast cancer cell.

In another embodiment, the present invention provides a method forinducing cell death in a target cell comprising the step of contactingor exposing the target cell to a composition comprising an effectiveamount of the immunotoxin complex of the invention. Cell death includesapoptotic cell death or necrotic cell death.

In another embodiment, the invention provides a method for treating asubject afflicted with ErbB-1 associated disease such as but not limitedto inflammatory bowel disease, comprising the step of administering tothe subject a pharmaceutical composition comprising an effective amountof the immunotoxin complex of the invention. In another embodiment, theinvention provides a method for treating a subject afflicted with ErbB-1associated cancer, comprising the step of administering to the subject apharmaceutical composition comprising an effective amount of theimmunotoxin complex of the invention. In another embodiment, treatingcancer is eliminating cancer cells by inducing cell death. In anotherembodiment, treating cancer is reducing tumor size. In anotherembodiment, treating cancer is inhibiting tumor growth. In anotherembodiment, treating cancer is reducing the risk of secondary tumors. Inanother embodiment, treating cancer is inhibiting metastasis.

In another embodiment, ErbB-1 associated cancer is lung cancer, analcancer, glioblastoma multiforme, epithelial cancer, prostate cancer,pancreatic cancer, head and neck cancer, breast cancer, ovarian cancer,and renal cancer.

In another embodiment, an effective amount or a therapeuticallyeffective amount of the immunotoxin is in the range of 0.05 μg/g (bodyweight)-10 μg/g (body weight). In another embodiment, an effectiveamount or a therapeutically effective amount of the immunotoxin is inthe range of 0.1 μg/g (body weight)-1 μg/g (body weight). In anotherembodiment, an effective amount or a therapeutically effective amount ofthe immunotoxin is in the range of 0.2 μg/g (body weight)-0.5 μg/g (bodyweight).

Exposure of the target cells to the immunotoxin complex may be carriedout by any of a number of routes, including without limitation,intravenous, intraperitoneal, subcutaneous, intramuscular andintralymphatic. As described herein, the complex may be one in which theantibody and the fusion protein are covalently associated.

The conjugates and complexes provided herein are useful in the treatmentand prevention of various diseases, syndromes and disorders, including,but not limited to: tumors, such as melanoma, ovarian cancer,neuroblastoma, pterygii, secondary lens clouding and the like andautoimmune diseases.

As used herein, the term “tumor-associated antigen” refers to an antigenwhich is found on or expressed by a tumor cell, but which also may befound on or expressed by other non-cancerous cells, for example, atover-expressed or developmentally untimely levels. A tumor-associatedantigen encompasses those associated with solid tumors and fluid tumorssuch as ascites fluid produced by ovarian cancer, pleural effusionproduced by lung carcinomas, and nonsolid hematologic tumors. The term“tissue-specific” refers to an antigen which is found mainly on aparticular tissue type.

The term “effective amount” as used herein means that amount of apharmaceutical agent or composition necessary to achieve the desiredspecific effect, for example against a target cell and/or inamelioration of a specific disease state.

As used herein, “treatment” means any manner in which the symptoms of acondition, disorder or disease are ameliorated or otherwise beneficiallyaltered. Treatment also encompasses any pharmaceutical use of thecompositions herein. As used herein, “amelioration” of the symptoms of aparticular disease or disorder refers to any lessening, whetherpermanent or temporary, lasting or transient, that can be attributed toor associated with administration of the composition.

In one embodiment, the immunoconjugates comprising the polynucleotideconstruct of the present invention may be used to treat tumors. In thesediseases, cell growth is excessive or uncontrolled. In one embodiment,the immunoconjugates may be used to treat tumors. In these diseases,cell growth is excessive or uncontrolled. Tumors suitable for treatmentwithin the context of this invention include, but are not limited to,breast tumors, gliomas, melanomas, prostate cancer, hepatomas, sarcomas,lymphomas, leukemias, ovarian tumors, thymomas, nephromas, pancreaticcancer, colon cancer, head and neck cancer, stomach cancer, lung cancer,mesotheliomas, myeloma, neuroblastoma, retinoblastoma, cervical cancer,uterine cancer, and squamous cell carcinoma of skin. For suchtreatments, ligands such as ErbB-1 can be chosen to bind to cell surfacereceptors that are generally preferentially expressed in tumors (e.g.MUC-1 and ErbB2). Through delivery of the compositions of the presentinvention, unwanted growth of cells may be slowed or halted, thusameliorating the disease. The methods utilized herein specificallytarget and kill or halt proliferation of tumor cells having receptorsfor the ligand on their surfaces. Treatment according to the methodsutilized herein result in killing or halting the proliferation of tumorcells expressing ErbB-1 receptor.

The immunoconjugate can be administered to a subject in any form or modewhich makes the compound bioavailable in effective amounts, includingoral and parenteral routes. For example, immunoconjugates can beadministered orally, parenterally, subcutaneously, intravenously,intramuscularly, transdermally, intraperitoneally, intralesionally,nasally, rectally and the like. One skilled in the art of preparingformulations can readily select the proper form and mode ofadministration depending upon the particular characteristics of thecompound selected, the disease state to be treated, the stage of thedisease, and other relevant circumstances.

The immunoconjugate can be administered alone or in the form of apharmaceutical composition in combination with pharmaceuticallyacceptable carriers or excipients, the proportion and nature of whichare determined by the solubility and chemical properties of the compoundselected, the chosen route of administration, and standardpharmaceutical practice. The compounds of the invention, while effectivethemselves, may be formulated and administered in the form of theirpharmaceutically acceptable acid addition salts for purposes ofstability, convenience of crystallization, increased solubility and thelike.

The pharmaceutical compositions comprising the immunoconjugate areprepared in a manner well known in the pharmaceutical art. The carrieror excipient may be a solid, semi-solid, or liquid material which canserve as a vehicle or medium for the active ingredient. Suitablecarriers or excipients are well known in the art. The pharmaceuticalcomposition may be adapted for oral or parenteral use and may beadministered to the subject in the form of tablets, capsules,suppositories, solution, suspensions, or the like.

The pharmaceutical compositions comprising the immunoconjugate may beadministered orally, for example, with an inert diluent or with anedible carrier. They may be enclosed in gelatin capsules or compressedinto tablets. For the purpose of oral therapeutic administration, thecompounds may be incorporated with excipients and used in the form oftablets, troches, capsules, elixirs, suspensions, syrups, wafers,chewing gums and the like. These preparations should contain at least 1%of the compound of the invention, the active ingredient, but may bevaried depending upon the particular form and may conveniently bebetween 1% to about 70% of the weight of the unit. The amount of thecompound present in compositions is such that a suitable dosage will beobtained. Preferred compositions and preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween 1.0-300 milligrams of the immunoconjugate.

The tablets, pills, capsules, troches and the like may also contain oneor more of the following constituents: binders such as microcrystallinecellulose, gum tragacanth or gelatin; excipients such as starch orlactose; disintegrating agents such as alginic acid, Primogel™, cornstarch and the like; lubricants such as magnesium stearate; glidantssuch as colloidal silicon dioxide; sweetening agents such as sucrose orsaccharin; flavoring agents such as peppermint, methyl salicylate ororange flavoring. When the dosage unit form is a capsule, it maycontain, in addition to materials of the above type, a liquid carriersuch as polyethylene glycol or fatty oil. Other dosage unit forms maycontain other various materials which modify the physical form of thedosage unit, for example, as coatings. Thus, tablets or pills may becoated with sugar, shellac, or other enteric coating agents. Syrup maycontain, in addition to the present compounds, sucrose as a sweeteningagent and certain preservatives, dyes and colorings and flavors.Materials used in preparing these various compositions should bepharmaceutically pure and non-toxic in the amounts used.

For the purpose of parenteral therapeutic administration, theimmunoconjugate of the present invention may be incorporated into asolution or suspension. These preparations should contain at least 0.1%of the immunotoxin of the invention, but may be varied to be between 0.1and about 50% of the weight thereof. The amount of the immunotoxinpresent in such compositions is such that a suitable dosage will beobtained. Preferred compositions and preparations according to thepresent invention are prepared so that a parenteral dosage unit containsbetween 1.0 to 100 milligrams of the immunotoxin.

The solutions or suspensions may also include the one or more of thefollowing constituents: sterile diluents such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerin, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl paraben; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylene diaminetetraacetic acid;buffers such as acetates, citrates or phosphates; and agents for theadjustment of tonicity such as sodium chloride or dextrose. Theparenteral preparation can be enclosed in ampules, disposable syringesor multiple dose vials made of glass or plastic.

According to yet another aspect, the invention provides a method forassessing whether an antibody specific for a cell surface antigen isinternalized into target cells expressing the antigen, the assaycomprising:

a. providing a recombinant fusion protein, wherein the fusion proteincomprises a first segment which is the ZZ Fc-binding domain derived fromS. aureus protein A and a second segment which is PE3 8 derived fromPseudomonas exotoxin;

b. selecting at least one antibody specific for the cell surface antigenpresent on the target cells;

c. combining the antibody from (b) with the ZZ-PE38 fusion protein from(a) to form a complex; and

d. exposing said target cells to the complex of (c); and

e. assessing whether the complex is internalized into the target cells.

In this method, the fusion protein may be linked to a detectable moiety,such as a chromophore, a fluorescent label or a radiolabel, as is knownin the art, in order to facilitate detection of internalization. Thismethod employs the fusion protein as a “general purpose” reagent forlarge scale screening of candidate antibodies for their potential use ascomponents of immunotoxins for targeted cell killing.

EXAMPLES Materials and Methods

All chemicals used were of analytical grade and were purchased fromSigma (Israel). Unless stated otherwise, reactions were carried out atroom temperature (about 25° C.). Unless stated otherwise, all thesecondary antibodies (HRP or fluorescently-labeled) used were fromJackson Immunoresearch Laboratories, USA.

Construction of Mammalian IgH and IgL Expression Vectors and Cloning ofImmunoglobulin H23 and FRP5 V genes to be Expressed as Chimeric IgG1Derivatives

The heavy chain expression vector pMAZ-IgH was constructed on thebackbone of pCMV/myc/ER/Neo (Invitrogen, USA). The human gamma 1constant heavy chain region (CH1-CH3) was recovered by PCR from humanlymphoid cDNA (Azriel-Rosenfeld et al 2004, J Mol Biol 335, 177-92)),using primers Hum-CH1-NheI-BACK (SEQ ID NO: 11) andHum-CH3-Stop-XbaI-FOR (SEQ ID NO: 12; Table 1). After sequencevalidation, the human constant fragment was inserted via NheI/XbaIrestriction sites into plasmid pCMV/myc/ER that had been linearized bythe same enzymes, resulting in the removal of the myc-tag and the ERretention signal of pCMV/myc/ER leaving only the intrinsic signalpeptide sequence. The murine H23 VH domain, derived from the anti MUC1recombinant scFv form was amplified from plasmid pMAZ1-MuH23 (Mazor etal 2005, Mol Immunol 42, 55-69) using primers H23-VH-BssHII-BACK (SEQ IDNO: 13) and H23-VH-NheI-FOR (SEQ ID NO:14; Table 1). The PCR product wasdigested and introduced into the heavy chain vector as a BssHII/NheIfragment, the resulting vector was named pMAZ-IgH-H23 (SEQ ID NO:7).

The light chain expression vector pMAZ-IgL was constructed on thebackbone of pcDNA3.1/Hygro (Invitrogen, USA). The plasmid DNA sequencebetween Sspl and XbaI sites was replaced by an SspI/XbaI fragmentrecovered from plasmid pCMV/myc/ER introducing the latter multiplecloning site containing recognition for BssHII and BsiWI restrictionenzymes to the resulting plasmid. Murine H23 light chain, derived fromthe anti MUC1 recombinant scFv form was amplified from plasmidpMAZ1-MuH23 (Mazor et al., 2005) using primers H23-VK-BssHII-BACK (SEQID NO: 15) and H23-CL-STOP-XbaI-FOR (SEQ ID NO: 16; Table 1) andintroduced into the light chain vector as BssHII/XbaI fragment, theresulting vector was named pMAZ-IgL-H23 (SEQ ID NO:8). All constructswere validated by DNA sequencing. The anti-ErbB2 FRP5 VH and VL domainwere cloned into mammalian expression vectors as followed, DNA ofplasmid pHEN1-FRP5(Fv) (Benhar et al., 2000) served as template in twoPCR reactions. The VH region was amplified using primers FRP5-VH-BACK(SEQ ID NO: 17) and FRP5-VH-BACK FOR (SEQ ID NO:18; Table 1) with theformer introducing BssHII site and the later NheI restriction site. ThePCR product was inserted into plasmid pMAZ-IgH via BssHII/NheI sites.The resulting plasmid was named pMAZ-IgH-FRP5 (SEQ ID NO:9).

TABLE 1 Primers Oligonucleotide Sequence Hum-CH1-NheI- 5′- BACKCCACAGGCGCGCACTCCGAGGTCCAACTGCAGGC SEQ ID NO: 11TAGCACCAAGGGCCCATCGGTC-3′ Hum-CH3-Stop-5′-TGTGTGTCTAGATTATTTACCCGGGGACAGG XbaI-FOR G-3′ SEQ ID NO: 12H23-VH-BssHII- 5′-CCACAGGCGCGCACTCCGAAGTGAAGCTTGA BACK GGAGTCTGG-3′SEQ ID NO: 13 H23-VH-NheI-FOR 5′-CTTGGTGCTAGCCGAAGAGACAGTGACCAGASEQ ID NO: 14 GT-3′ H23-VK-BssHII- 5′-CCACAGGCGCGCACTCCCAGCTCCAGATGACBAC CCAGTC-3′ SEQ ID NO: 15 H23-CL-STOP-5′-TCTCTCTCTAGATTAACACTCTCCCCTGTTG XbaI-FOR AAGC-3′ SEQ ID NO: 16FRP5-VH-BACK 5′-CCACAGGCGCGCACTCCCAGGTACAACTGCA SEQ ID NO: 17GCAGTCTGG-3′ FRP5-VH-FOR 5′-CTTGGTGCTAGCAGAGGAAACGGTGACCGTGSEQ ID NO: 18 GTCC-3′ FRP5-VK-BACK 5′-CCACAGGCGCGCACTCCCGACATCCAGCTGCSEQ ID NO: 19 CCAGTC-3′ FRP5-VK-FOR 5′-AGCCACCGTACGTTTGATCTCCAATTTTGTCSEQ ID NO: 20 CCCCGAGC-3′ ZZ-Nco-FOR 5-CCGCTTCCATGGTAGACAACAAATTCAACAAASEQ ID NO: 21 G-3′ ZZ-Not-REV 5′-GGGTTTAGCGGCCGCTTTCGGCGCCTGAGCASEQ ID NO: 22 TCATTTAG-3′

The FRP5 V_(K) region was amplified using pHEN1-FRP5(Fv) (Benhar et al.,2000) as template with primers FRP5-VK-BACK/FOR ((SEQ ID NOS: 19 and 20respectively), introducing restriction sites BssHII at the 5′ end andBsiWI at the 3′ end, respectively. The PCR product was digested withrestriction enzymes BssHII and BsiWI and cloned into plasmid pMAZ-IgLthat had been linearized by the same enzymes. The resulting plasmid wasnamed pMAZ-IgL-FRP5 (SEQ ID NO:10).

Cell Lines

Cell lines used were the human breast adenocarcinoma SKBR3 cell line,the human epidermoid carcinoma A431 cell line, the human breastcarcinoma T47D and MCF7 cell lines, the human mammary carcinomaMDA-MB231 cell line and the human kidney HEK293 cell line. All celllines were maintained in Dulbecco's modified Eagle medium (DMEM)containing 10% fetal calf serum (FCS), 2 μg/ml blasticidin (Invitrogen),penicillin, and streptomycin, unless specified otherwise. Additionally,the human head and neck H&N(SCC-1 and SCC-9) and prostate (C1-1 clone 3and clone 70 (19) cancer cell lines as well as human foreskinfibroblasts were used.

The cells were cultured routinely in DMEM supplemented with 10%heat-inactivated fetal calf serum (FCS), antibiotics, glutamine, 0.1 mMnon-essential amino acids, 1.0 mM sodium pyruvate (BiologicalIndustries, Beit HaEmeq, Israel), and grown in 5% CO₂ at 37° C. in awater jacketed incubator in humid atmosphere. The cells were harvestedby trypsin solution with 1-2 passages per week in a split ratio of1:3-5. The 24-hrs cell cultures were used in all the experiments.

Transfection of HEK293 Cells with Mammalian pMAZ-IgH and pMAZ-IgLExpression Vectors and Screening of Cell Culture Supernatants of StableClones Expressing Chimeric IgG1 Derivatives using Dot-Blot Analysis

Co-transfections of HEK293 cells with pMAZ-IgH and pMAZ-IgL expressionvectors (for either chimeric H23 IgG, chH23, or for chimeric FRP5 IgG,chFRP5) were performed using FUGENE 6 nonliposomal transfection reagent(Roche, Brussels, Belgium) according to the manufacturer's instructions.Briefly, 10⁶ cells were seeded into 6 well plates and 24 hours aftertransfection, limiting dilutions were performed into 96-well platesmedium containing 1.2 mg/ml of G418 and 200 μg/ml of hygromicin at aratio of 1000 cells/well. Supernatants of single colonies were grown tonear confluence on medium containing selection markers were tested forIgG1 secretion by dot-blot analysis and analyzed in ELISA for antibodybinding to the corresponding antigen (MUC1 or ErbB2). To screen for IgGproducing cells, 100 μl supernatants of stable clones were applied via avacuum manifold onto a nitrocellulose filter using a dot-blot apparatus(Schleicher & Schuell. USA). After blocking the membrane with 5% (v/v)non-fat milk in Tris Buffered Saline (TBS) for 1 h at room tmperature,the membrane was washed briefly with TBS and incubated withHRPconjugated goat anti human antibodies (×10,000 dilution in TBS/2%milk) for 1 h at room temperature. The membrane was developed using theRENAISSANCE Western blot Chemiluminescence Reagent (NEN, USA) accordingto the supplier's instructions. To determine the amount of chimeric IgGsecreted from positive clones, 100 μl supernatants were applied in atwo-fold dilution series via a vacuum manifold onto a nitrocellulosefilter using a dot-blot apparatus alongside a two-fold dilution seriesof commercial humane IgG standard (Jackson ImmunoRsearch Laboratories,USA), starting with a concentration of 1000 ng/ml. After blocking themembrane with 5% (v/v) non-fat milk in TBS for 1 h at room temperature,the membrane was washed briefly with TBS and incubated withHRP-conjugated goat anti human antibodies (×10,000 dilution in TBS/2%milk) for 1 h at room temperature. The membrane was developed asdescribed above.

Evaluation of IgG Producing Clones for MUC1-Binding by ELISA

IgG producing clones were tested for MUC1-binding as follows: ELISAplates were coated with a 10-fold dilution of conditioned medium ofmouse DA3 cells transfected with a secreted isoform of MUC1 diluted in50 mM NaHCO₃ buffer (pH 9.6) at 4° C. for 20 h and blocked with 2% (v/v)non-fat milk in PBS for 2 h at 37° C. essentially as described (Mazor etal 2005, Mol Immunol 42, 55-69). All subsequent steps were done at roomtemperature. 100 μl supernatants of IgG1 producing clones were appliedonto the plates and incubated for 1.5 h (diluted 1:2 in 2% (v/v) non-fatmilk/PBS). Following incubation the plates were washed ×3 with PBST.Bound IgG was detected with HRP-conjugated goat anti human antibodies(×10,000 dilution in PBST). The ELISA was developed using thechromogenic HRP substrate TMB (Sigma, Israel) and color development wasterminated with 1 M H₂SO₄. The plates were read at 450 nm.

Production and Purification of Chimeric H23 IgG1 from Culture Media ofStable Transfected HEK293 Cells

Approximately 3×10⁶-transfected HEK293 cells (chH23 clone B2 or chFRP5clone G1) were cultured in 75 cm² flasks containing DMEM medium (BeitHaemek, Israel), supplemented with 10% fetal calf serum, 1.2 mg/ml G418and 200 μg/ml hygromycin, at 37° C., 5% CO₂, in a humidified incubator.The culture was allowed to grow to 80% confluence followed by a gradualstarvation of the cells to fetal calf sera (FCS), in a two-fold dilutionseries (reduction in serum concentration) for a period of 24 h of eachdilution. The cells were totally deprived of FCS 72 h prior toharvesting. Chimeric IgGs were purified using protein A-SEPHAROSE(Amersham Biosciences, Sweden) chromatography. Briefly, 250 ml ofculture supernatant was diluted 1:1 with loading buffer (20 mM Na₂HPO₄,2 Mm NaH₂PO₄) and loaded onto a 5-ml protein A column at a flow rate of2 ml/min. The column was extensively washed with loading buffer. Boundchimeric IgG was eluted with 0.1 M of citric acid (pH 3) and neutralizedwith 1 M Tris/HCl (pH 9). Protein-containing fractions were combined,dialyzed against 5 liter PBS (16 h, 4° C.), sterile filtered and storedat 4° C. Purified chimeric IgGs were analyzed by 12%/SDS polyacrylamidegel electrophoresis under reducing conditions and stained with GELCODEBLUE® dye (Pierce, USA). For Western blot, purified IgGs were separatedby 12%/SDS polyacrylamide gel electrophoresis under reducing conditionsand electro-transferred onto nitrocellulose membrane. Chimeric IgGs weredetected with HRP-conjugated goat anti human antibodies. The membranewas developed using the RENAISSANCE Western blot ChemiluminescenceReagent (NEN, USA) according to the supplier's instructions.

Construction of pET22b-ZZ-PE Expression Vector

Plasmid pET22b-ZZ-PE38 was designed to allow the expression of solubleZZ-Pseudomonas exotoxin A (PE38) fusion protein secreted to theperiplasm of BL-21 (DE3) E. coli cells.

Plasmid pB1(Fv)-PE38 (Benhar and Pastan 1995, Clin Cancer Res 1,1023-1029) carries the scFv of anti Le^(Y) monoclonal antibody B1 fusedto a truncated fragment of Pseudomonas exotoxin A. As all members ofthat vector series (Brinkmann et al., 1991), the single-chain Fvfragment (scFv) is cloned between NdeI and HindIII sites. These siteswere changed to NcoI and NotI sites, respectively, using site-directedmutagenesis essentially as described (Benhar et al 1994, J Biol Chem269, 13398-404). The resulting plasmid was named pIB98-NN-B1(Fv)-PE38.The scFv-PE38 coding DNA fragment was recovered frompIB98-NN-B1(Fv)-PE38 by digestion with NcoI and EcoRI and ligated into avector fragment isolated from pET22b (Novagen, USA) by digestion withthe same enzymes. The resulting plasmid was named pET22b-NN-B1(Fv)-PE38.

Next, the scFv cloned in pET22b-NN-B1(Fv)-PE38 was replaced by anNcoI-NotI fragment of the ZZ domain that was isolated using pDS-ZZplasmid DNA as template in a PCR reaction with primers ZZ-Nco-FOR (SEQID NO: 21) and ZZ-Not-BACK (SEQ ID NO: 22; Table 1). In the resultingplasmid, pET22b-ZZ-PE38, a T7 promoter controls the expression of acassette comprising a pelB leader for secretion followed by theZZ-domain-PE38 fusion.

Expression and Purification of ZZ-PE38 Fusion Protein

Periplasmic production of soluble ZZ-PE38 fusion protein was performedon a 1 liter scale. E. coli BL21 (DE3) cells transformed withpET22b-ZZ-PE38 expression vector were grown in 1 liter of SB mediumsupplemented with 100 μg/ml ampicillin, 0.5% (w/v) glucose and 0.4 gr/lMgSO₄ at 37° C. When the cells reached A₆₀₀ of 2.5 they were induced forprotein overexpression with 1 mM IPTG at 30° C. for 3 h. Followedinduction, the cells were collected by centrifugation (15 min, 4000 rpmat 4° C., Beckman GS3 rotor) and the periplasmic fractions were preparedby gently resuspending the cell pellet using glass beads in 200 ml ofice-cold 20% sucrose, 30 mM Tris-HCl (pH 7.4), 1 mM EDTA and left on icefor 15 min. Next, cells were collected by centrifugation as describedabove, the sup was discarded off and the culture was gently re-suspendedin 200 ml of ice cold sterile water and left on ice for 15 min.Following incubation on ice, the periplasmic fraction was obtained bycollecting the cells by centrifugation (15 min, 7000 rpm at 4° C.,Beckman rotor GSA) and collecting the supernatant. The resultingsupernatant (periplasmic fraction) was adjusted to 20 mM Tris-HCl (pH7.4), 1 mM EDTA and the ZZ-PE38 fusion protein was purified from theperiplasmic extract in two sequential chromatography steps ofQ-SEPHAROSE and MONO-Q anion exchange columns (Pharmacia LKB) using fastprotein liquid chromatography (FPLC), (Pharmacia, Sweden).

Preparation of chIgG-ZZ-PE38 Immunoconjugates

Conjugation of chimeric IgG1 antibodies with the ZZ-PE38 fusion proteinwas performed as follows, 0.5 ml of 4.5 mg/ml chH23 or chFRP5 in PBSwere mixed with 0.5 ml of 4.5 mg/ml ZZ-PE38 fusion protein in PBS (3fold molar excess of ZZ-PE38 over chimeric IgG) in an Eppendorf tubehead-over-head for 16 h at 4° C. Separation of excess ZZ-PE38 fromchIgG-ZZ-PE38 complex was performed by applying the sample to a SUPERDEX75 size-exclusion column (Pharmacia LKB) using fast protein liquidchromatography (FPLC), (Pharmacia, Sweden). The proteins were separatedin PBS at a flow rate of 1 ml/min. 1 ml fractions were collected.

Crosslinking of chFRP5-ZZ-PE38 immunoconjugate using the BS³ reagent(Pierce, USA) was performed according to the supplier's recommendations.Briefly, 100 μl of 3 mg/ml chFRP5-ZZ-P38 in PBS were mixed with a20-fold molar excess of BS³ solution (dissolved in PBS). The preparationwas incubate for 2 h at 4° C. Excess of non-reacting BS³ reagent wasremoved by dialysis against 1 liter of PBS buffer using SLIDE-A-LYZER®Dialysis cassette with 3,500 MW cutoff at 4° C. for 16 hr with gentlestirring. Crosslinked-chFRP5-ZZ-PE38 in PBS buffer was sterile filteredand stored at 4° C.

Evaluation of Antigen Binding by ELISA

Analysis of binding to MUC1 by H23 was done as follows, ELISA plateswere coated with a 10-fold dilution of MUC1 transfected DA3 cellsconditioned medium diluted in 50 mM NaHCO₃ buffer (pH 9.6) at 4° C. for20 h and blocked with 2% (v/v) non-fat milk in PBS for 2 h at 37° C.,essentially as described (Mazor et al 2005, supra). All subsequent stepswere done at room temperature. 2 μg/ml of purified chimeric H23 (chH23)and murine H23 Mab were applied onto the plates in a two-fold dilutionseries. Following incubation the plates were washed ×3 with PBST.HRP-conjugated goat anti human and HRP-conjugated goat anti-mouseantibodies were used as secondary antibodies (×10,000 dilution in PBST).The ELISA was developed using the chromogenic HRP substrate TMB (Sigma,Israel) and color development was terminated with 1 M H₂SO₄. The resultswere plotted as absorbance at 450 nm and the apparent binding-affinitywas estimated as the IgG concentration that generates 50% of the maximalbinding signal.

Evaluation of Binding-Affinity by Whole-Cell ELISA

Cellular MUC 1 binding with chH23 IgG and murine H23 mAb was tested bywhole-cell ELISA as follows; cell MUC1 positive cells were the humanbreast carcinoma T47D cell line. Following trypsinization, cells werewashed once with 2% fetal calf serum, 0.05% NaN₃ in PBS (incubationbuffer). In each experiment approximately 10⁶ cells were divided intoindividual immunotubes (Nunc, Sweden). To confirm the specificity,antibodies (1 μg/ml) were incubated with or without an excess of MUC1protein from DA3-MUC1-transected cell line, for 1 h at room temperatureprior to incubation with the cells and then were added to the cell tubesfor 1.5 h at 4° C. After washing ×2 with incubation buffer,HRP-conjugated goat anti human and HRP-conjugated goat anti mouse(×2000) were added to the appropriate tubes for 1 h at 4° C. Detectionof cell bound antibodies was performed by addition of 0.5 ml of thechromogenic HRP substrate TMB (Sigma, Israel) to each tube and colordevelopment was terminated with 0.25 ml of 1 M H₂SO₄. Finally, the tubeswere centrifuged for 10 min at 4000 rpm and color intensity ofsupernatants was measured at 450 nm. The apparent binding-affinity wasestimated as the IgG concentration that generates 50% of the maximalbinding signal.

Cellular ErbB2-binding of chFRP5 IgG was evaluated by whole-cell ELISAperformed as described above using the breast cancer cell line SKBR3 asErbB2 positive.

Evaluation of cellular MUC1 binding activity of chH23-ZZ-PE38 orchFRP5-ZZ-PE38 immunotoxin by whole-cell ELISA was performed essentiallyas described above. Rabbit anti-PE sera (×200 dilution; Kindly providedby Dr. Ira Pastan, NCl, NIH) mixed with HRP-conjugated goat anti-rabbit(×1500 dilution) were used for the detection of bound chH23-ZZ-PE38 oror chFRP5-ZZ-PE38 immunotoxins.

Flow Cytometry

Cellular MUC1 binding with chH23 IgG and murine H23 mAb was tested byflow cytometry. Cell lines used were the mouse cell line DA3, theMUC1-transfected cell line DA3-MUC1 (Baruch et al 1999, Cancer Res 59,1552-61) the breast carcinoma lines T47D and MCF7 and the human kidneycell line HEK293. Approximately 5×10⁵ cells were used in eachexperiment. After trypsinization, cells were washed once in 2% fetalcalf serum, 0.05% NaN₃ in PBS (FACS buffer). To confirm the specificity,antibodies (10 μg/ml) were incubated with or without an excess of MUC1protein from DA3-MUC1-transected cell line, for 1 h at room temperatureprior to incubation with the cells and then were added to the cell tubesfor 1 h at 4° C. After washing ×2 with FACS buffer, FITC-labeled goatanti human and FITC-labeled goat anti mouse (×50 dilution) was added tothe appropriate tubes for 45 min at 4° C. Detection of bound antibodieswas performed by means of flow cytometry on a FACS-CALIBUR (BectonDickinson, Calif.) and results were analyzed with the CELLQUEST program(Becton Dickinson).

Evaluation of cellular MUC1 binding activity of chH23-ZZ-PE38immunotoxin was tested by flow cytometry essentially as described above.To confirm specificity, chH23-ZZ-PE38 immunoconjugate was incubated inthe presence or absence of 10-fold excess of un-conjugated chH23 IgG1prior to incubation with the cells. Rabbit anti-PE sera (×50 dilution)mixed with FITC-labeled goat anti-rabbit (×50 dilution) were used forthe detection of bound chH23-ZZ-PE38 immunotoxin.

Evaluation of cellular ErbB2-binding by chFRP5 IgG was performed asdescribed above. Cells expression various levels of ErbB2 on theirsurface were tested.

Anti-EGFR Antibody

Chimeric monoclonal anti-EGFR antibody—cetuximab (Erbitux®)—waspurchased from Merck (Germany). Human IgG was provided by Jacksonimmuno-research (US). Goat-anti-mouse HRP antibody used as a secondaryantibody was purchased from Santa Cruz (US).

Western Blot.

The cells were grown in 10-cm tissue culture dishes, washed briefly withice-cold PBS and treated for 30 minutes on ice with lysis buffercontaining 50 mM HEPES pH 7.5, 150 mM NaCl, 1% Triton X-100, 10%glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 30 mM b-glycerol phosphate, 2mM sodium ortho-vanadate, 2 mM PMSF and proteinase inhibitors (10 μg/mlaprotinin and protein inhibitor cocktail). Cell lysates were cleared bybrief centrifugation (14,000 rpm, 1 min) and then kept at −80° C. untiluse. Protein measurement was performed using the BCA Protein Assay Kit(Pierce) with bovine serum albumin as a standard. After electrophoresis,the proteins were electrophoretically transferred to nitrocellulosemembranes (1-2 hr, 4° C.) and then saturated overnight at roomtemperature in a blocking solution (TBST 50 mM Tris-HCl, pH 7.5, 150 mMNaCl and 0.1% Tween 20 with the addition of 5% low fat milk). Themembranes were then incubated for 1.5 hr at room temperature with theprimary antibody—rabbit polyclonal IgG (purchased from Sigma) diluted inTBST containing 2% low fat milk. The membranes were washed extensivelywith TBST solution and incubated for 1 hr at room temperature withconjugated goat anti rabbit IgG-HRP (from Santa Cruz, Calif.). Signalswere detected by using the enhanced chemo-luminescence method (EZ-ECL,Biological Industries, Beit Haemek, Israel). The band density wasanalyzed by TINA (2.0 ver.) software.

Anti-EGFR ELISA

The cells were grown in 96 micro-well flat-bottom plates to fullconfluence over a period of 24-48 hrs. The cells were than washed withPBS and fixed with 3% formaldehyde in PBS for 5 min at −20° C. The cellswere washed with PBS and then incubated for 1 hr with monoclonalanti-EGFR antibody—cetuximab used as primary antibody. The next step wascell washing with PBS to remove unbound primary antibody and then theincubation was performed for 1 hr with goat-anti-human. HRP antibodyused as a secondary antibody and the color reaction was developed usingthe HRP substrate TMB. The cells were washed with PBS. The plates wereplaced on a mechanical plate shaker of a computerized automaticmicro-well plate spectrophotometer (Sunrise, Switzerland) and shaken for30 sec. The optical densities (OD₄₅₀) of the dye were read at 450 nm.

Fluorescent Microscopy-Binding of Cetuximab

Confocal microscopy was implemented to test the binding of cetuximab tothe cells. The cells were seeded on cover slips placed in a 6 wellplate. The cells were allowed to grow for 2 days and then fixed withice-cold methanol and acetone. The cells were incubated withcorresponding primary antibodies for 2 hrs at room temperature andwashed with PBS. Secondary (photoreactive) antibodies were added closelyto microscopy.

Flow Cytometry (FACS)

Cells were harvested (˜10⁶ cells/ml), centrifuged at 4° C. for 10 min at1,500 rpm, fixed in ice-cold methanol for 15 min and then washed withPBS. The fixed cells were incubated with cetuximab for 2 hrs and thenwith anti-human FITC conjugated antibodies for 1 hr. The cells wereanalyzed by FACScan (Becton, Dickinson). The data were evaluated usingwin MDI (2.9 ver.) software. The comparison of the cells tested wasperformed by the peak of fluorescence of stained cells.

Colorimetric Tetrazolium Salt (XTT) Assay for Cell Survival

Cell density was evaluated by colorimetric assay based on the conversionof XTT to orange-colored formazan compounds by cellular dehydrogenases.The advantage of this assay with the use of 96 micro-well plates is inthe ability to rapidly test numerous arms of each experiment in the sameconditions. Typically, 200 μl with a known number of cells fromexponentially growing cultures were plated in 96 micro-well flat-bottomplates. To determine the effect of the treatment tested on cellsurvival, the therapy was started following 24 hr of cell culturing. Theagents tested were added in varying concentrations to each of threereplicate wells and incubated for 4 days. The effect on cell survivalwas calculated by comparing the density of intact cells to the treatedcells determined by XTT assay (Biological Industries, Beit Haemek,Israel). A freshly prepared mixture of XTT and an activation reagent(PMS) was added into each well (50 μA). Following 2 hrs of incubation at37° C., the OD₄₅₀ was recorded as described in the ELISA section. Themeasurements were repeated following 4 and 6 hrs of incubation. The timepoint of the assay with optimal OD readings was chosen to assay the cellnumber. When more than one time point fitted these criteria, the resultsin the different time points were normalized and averaged. The ODreadings were shown to correlate well (r>0.97-0.99) with the number ofseeded cells/well.

Animals.

The 4-6-week-old male athymic CD-1 nude mice were obtained from Harlan(Jerusalem, Israel). The animals were housed in a laminar flow cabinetunder pathogen-free conditions in standard vinyl cages with air filtertops. Cages, bedding and water were autoclaved before use. The localEthics Committee for Accreditation of Laboratory Animal Care approvedall facilities in accordance with the current regulations and standardsof the Israeli Ministry of Health.

Ectopic Model for Tumor Growth

H&N cancer cells—SCC-1 (3×10⁶ cells/0.2 ml saline) were injectedsubcutaneously (s.c.) into both flank area of the mice. The progressionof tumors as well as the effect of the treatment tested was followed upby the measurement of the subcutaneous tumors' size 2 times per weektill the end of the experiment. Tumor volume was determined by directmeasurement of tumor with digital caliper and calculated by the formula:v=a (large diameter)×b² (small diameter²)×π/6. The experiment wasstopped when the mice have become moribund or when the tumors havereached 2 cm in their widest diameter or when the weight of the micehave decreased by about 40% according to the regulations of theInstitutional Committee of Animal Welfare. Developed tumors underwentpathologic evaluation at the Pathology Department of Tel-Aviv SouraskyMedical Center.

Treatment of Tumor Bearing Mice

Treatment of the tumor bearing mice was started 2 weeks after cellimplantation when tumor volume was about 100 mm³. The mice wererandomized according tumor size in 5 groups relative to treatmenttested. All tested agents (200 μl/mouse) were injected intra-peritonealtwice weekly: ZZ-PE38 alone (0.20 μg/g; 4 μg/mouse); cetuximab alone(0.40 μg/g; 8 μg/mouse); cetuximab-ZZ-PE38 complex (0.25 μg/Kg; 5μg/mouse) and cetuximab-ZZ-PE38 complex (0.50 μg/g; 10 μg/mouse).Overall, six treatments have been given during 3 weeks and then the micewere followed up to 56 days from tumor cell injection.

Immunohistochemistry

A large set of paraffin-embedded normal and tumor tissue sections wereused to compare immunohistochemical staining obtained by the purifiedchH23 IgG and the murine H23 mAb. Five-micrometer sections ofparaffin-embedded tissues were deparafinized with xylene and ethanol andrehydrated with hyaluronidase (Fiorentini et al 1997, Immunotechnology3, 45-59). Endogenous peroxidase was blocked with 0.3% H₂O₂ in methanoland slides were preincubated with 10% goat serum in PBS for 30 min.Antibodies were diluted to a concentration of 20 μg/ml in PBS containing10% goat serum and incubated with slides overnight at 4° C. Afterrinsing with PBS, HRP-conjugated goat anti human and HRP-conjugated goatanti mouse (1/50 dilution) was added to the appropriate slides for 30min at room temperature. After each incubation step, slides were washedfor 10 min with PBS. Staining was performed using the HRP chromogenicsubstrate 3,3′-diaminobenzidine (Sigma, Israel), followed bycounterstaining with hematoxylin.

A large set of adjacent Paraffin-embedded tumor tissue sections wereused to compare immunohistochemical staining obtained by the purifiedchFRP5 IgG and of the two positive anti-ErbB2 Mab; Herceptin® and acommercial mouse anti-ErbB2 Mab. The sections were processed anddeveloped as described above.

Analysis of IgG Internalization using Confocal Microscopy

Internalization of chH23 IgG1 and of chFRP5 IgG was evaluated usingconfocal microscopy. The human breast carcinoma T47D cell line was usedto evaluate the internalization capabilities of chH23 at 4° C. and 37°C. The human breast carcinoma SKBR3 cell line was used to evaluate theinternalization capabilities of chFRP5 at 4° C. and 37° C. Sterile 24 mmcover slips were placed in a 6 well plate and incubated for 1 hr at roomtemperature with 800 μl of 10 μg/ml poly-L-lysine followed by two washeswith 1 ml PBS each. Approximately 4×10⁵ cells were seeded on the coverslips in each well and grown to 40%-50% confluence in DMEM supplementedwith 10% FCS (complete medium). Cells tested for chH23 internalizationat 4° C. were preincubated with complete medium supplemented with 0.5%NaN₃ for 2 h in 4° C. prior to the addition of the antibody (5 μg/ml)and incubation of 1 h in 4° C. Cells tested for chH23 internalization at37° C. were incubated with 5 μg/ml of the antibody in complete mediumfor 1 h at 37° C. in a humidified atmosphere of 95% air and 5% CO₂.Subsequent the incubation with the antibody, the cells were gentlywashed twice with PBS to remove excess mAb and incubated for 2 h underthe same conditions of the previous step. Next, the cells were gentlywashed twice with PBS and fixed in two sequential steps of incubationfor 10 min with ice cold methanol followed by 10 min incubation with icecold acetone. Following the fixation step, the cells were gently washedwith PBS and blocked with 10% normal goat serum (NGS) in PBS for 25 minat RT. The blocking solution was aspirated and FITC-labeled goat antihuman (×150 dilution) was added cells were incubated with ×150 dilutionof FITC conjugation anti human antibodies for 2 hr at RT. Finally, thecells were gently washed ×3 with PBS and staining pattern (membranous orintracellular) images were acquired using a LSM 510 laser scanningconfocal microscope (Vontz 3403B).

Cell-Viability Assay

The cell-killing activity of immunotoxins was measured by MTT assay.Target and control cells were seeded in 96-well plates at a density of10⁴ cells/well in DMEM supplemented with 10% FCS. Various concentrationof chH23-ZZ-PE38 and relevant control proteins were added in triplicateand the cells were incubated for 48 hr at 37° C. in 5% CO₂ atmosphere.After treatment, the media was replaced by immunotoxin-free media (125μl per well) containing 5 mg/ml MTT reagent (Thiazolyl Blue TetrazoliamBromide, Sigma, Israel, dissolved in PBS) and the cells were incubatedfor another 4 h. MTT-formazan crystals were dissolved by the addition of20% SDS, 50% DMF, pH 4.7 (100 μl per well) and incubation for 16 h at37° C. in 5% CO₂ atmosphere. Absorbance at 570 nm was recorded on anautomatized microtiter plate reader. It was established that opticaldensity was directly proportional to the cell number up to the densityreached by the end of the assay. Identical concentrations andcombinations were tested in three separate wells per assay and the assaywas performed at least three times. The results were expressed aspercentage of living cells in comparison to the untreated controls thatwere processed simultaneously using the following equation: (A₅₇₀ oftreated sample/A₅₇₀ of untreated sample) ×100. The IC₅₀ values weredefined as the immunotoxin concentrations inhibiting cell growth by 50%.

Functional Stability Analysis

The functional stability of chFRP5-ZZ-PE38 immunoconjugate and thecrosslinked-chFRP5-ZZ-PE38 derivative was determined by incubation ofthe purified proteins at 37° C. for varying periods under threeconditions; diluted in PBS, diluted in PBS containing a 10-fold molarexcess of commercial protein-A purified human IgG antibodies, anddiluted in 100% human serum. These incubations were followed by analysisof cellular ErbB2 binding of SKBR3 cells by whole-cell ELISA and incell-killing of A431 cells using MTT assay. At each time-point, analiquot was removed and spun for 10 min at 20,000 g to removeprecipitated protein before being analyzed for cellular ErbB2 bindingand cell-killing of A431 cells.

Pharmacokinetics of chFRP5-ZZ-PE38 and scFv(FRP5)-ETA in Mice Serum

The recombinant immunotoxin scFv(FRP5)-ETA was kindly provided by Prof.Winfried Wels, Georg Speyer Haus, Frankfurt am Main, Germany. This is arecombinant immunotoxin comprising the scFv derivative of FRP5 linked toa truncated Pseudomonas exotoxin (Wels et al 1992, Cancer Res 52,6310-7). The relevant toxin fragment is known in the US as PE40 (fromwhich PE38 was derived by deletion of a short segment of domain Ib)Brinkman et al 1991, Proc Natl Acad Sci USA 88, 8616-20) and in Europeit is referred to as ETA. To evaluate and compare the bloodpharmacokinetics of chFRP5-ZZ-PE38 and scFv(FRP5)-ETA, female BALB/cmice (6-8 weeks old, ˜20 g) were given a single i.v. dose of 15 μgchFRP5-ZZ-PE38 or 5 μg scFv(FRP5)-ETA diluted in 200 μl PBS by injectioninto the tail vein. Blood samples were collected from the orbital veinof mice injected with scFv(FRP5)-ETA at 2, 5, 10, 20, 30, 60, 120 and240 min after injection and at 2, 5, 10, 20, 30, 60, 120, 240, 480 and1440 min for mice injected with chFRP5-ZZ-PE38. Each mouse was bled twoor three times, so different mice were used to collect data for thevarious time points. Each time point represents the mean of resultsobtained from two mice. After clotting the blood samples on ice theserum concentration of chFRP5-ZZ-PE38 and scFv(FRP5)-ETA was determinedby dot-blot analysis and the concentration of remaining activeimmunotoxin was determined by incubating dilutions of the serum withA431 cells and measuring cell-viability by MTT assay. Determination ofimmunotoxins serum concentration by dot-blot analysis was performed asfollows, 100 μA of serum diluted 1:100 in PBS of each mouse at a timegroup was applied in a two-fold dilution series via a vacuum manifoldonto a nitrocellulose filter using a dot-blot apparatus (Schleicher &Schuell. USA), alongside a two-fold dilution series of 100 ng/ml ofchFRP5-ZZ-PE38 or scFv(FRP5)-ETA that were used to determine theimmunotoxin concentration in each serum sample. Rabbit anti-PE sera(×2500 dilution) mixed with HRP-conjugated goat anti-rabbit antibodies(×5000 dilution) were used for the detection of the immunotoxins. Themembrane was developed using the RENAISSANCE Western blotChemiluminescence Reagent (NEN, USA) according to the supplier'sinstructions. Determination of remaining active immunotoxinconcentration in serum sample by MTT assay was performed as follows, 100μl of serum diluted 1:100 in DMEM medium supplemented with 10% FCS ofeach mouse at a time group were added in a 10-fold dilution series toA431 cells seeded in 96-well plates at a density of 10⁴ cells/well. Astandard curve, obtained by incubating A431 cells alongside a serialdilutions of chFRP5-ZZ-PE38 or scFv(FRP5)-ETA was used to determine theimmunotoxin concentration in each serum sample. The MTT cell-viabilityassay was performed essentially as described above.

Toxicity Assays of ZZ-PE38 and ZZ-PE38 Immunoconjugates in Mice

The single-dose mouse LD₅₀ of ZZ-PE38 fusion protein and threeimmunoconjugates; chFRP5-ZZ-PE38, crosslinked-chFRP5-ZZ-PE38 andhIgG-ZZ-PE38 was tested using female BALB/c mice (6-8 weeks old, ˜20 g),that were given a single i.v. injection with different doses of eachprotein diluted in 200 μl PBS. Mice were monitored for weight loss ordeath for 2 weeks after injection.

Antitumor Activity of chFRP5-ZZ-PE38 and scFv(FRP5)-ETA

To establish xenografts, female BALB/c athymic nude mice (6-8 weeks old,˜20 g) 3-5 mice per group were injected i.v. with 1.5×10⁶ A431 cellssuspended in 0.2 ml PBS. By day 9 post injection, tumors of about 30-40mm³ had formed. Mice were treated on days 9, 12, 15, 18 and 21 by i.v.injections of different doses of the two immunotoxins diluted in PBS.Tumors were measured with a caliper at 3-day intervals, and the tumorvolumes were calculated according to the formula:volume=(length)×(width)²×(0.4). Animals were sacrificed when tumorsreached 2 cm in diameter or when animals appeared to be in distress.

Statistics

The results for each variant in the in vitro experiments wererepresented as an average of 2-4 experiments, and each arm was typicallyperformed in triplicate. Statistical analysis was performed using theGraphPadPrizm 5.0 software. The mean values and standard errors werecalculated for each point from the pooled normalized data. Cytotoxicitywas evaluated using non liner regression model of one phase decay andIC₅₀ was defined as the concentration at which 50% of the maximum cellkilling was obtained. The difference between the arms was analyzed usingtwo ways ANOVA. The results for the in vivo experiments were analyzed byANOVA and Tukey's Multiple Comparison Test with comparison of all pairsof the treatment tested. In all cases the difference was considered asstatistically significant if p<0.05.

Comparison in tumor size between different treatment arms was conductedat 3 days after end of treatment (day 35) and at the end of follow-up(day 56) using two tailed t-test with unequal variance.

Example 1 Construction of Mammalian Expression Vectors for Human IgG1Derivatives

Mammalian vector pMAZ-IgH for human γ1 heavy chain expression andpMAZ-IgL for human κ light chain expression were designed for productionof human IgG1 antibodies in mammalian cell culture (FIG. 1). Each vectorcarries the germline sequence of the respective heavy or light chaingene including its polyadenylation site located downstream of thetranslation termination codon. VH domains are introduced into the IgHexpression vector via the BssHII and NheI restriction sites, whereas, VLdomains are cloned into the IgL expression vector as BssHII and BsiWIfragments. The IgH plasmid carries a neomycine expression cassette forgeneticin (G418) selection, while the IgL plasmid carries a hygromycin Bresistance cassette for the isolation of stable transfectants. Thestrong human cytomegalovirus early promoter drives both the heavy andlight chain genes. In addition, both vectors contain an ampicillinselectable marker and SV40, ColE1 and f1 origin of replication.

The variable region genes encoding the murine anti-MUC1 H23 mAb werecloned for expression as chimeric γ1/kappa antibody into the mammalianpMAZ-IgH and pMAZ-IgL expression vectors (SEQ ID NOS: 7 and 8,respectively) as described in Materials and Methods. Afterco-transfection of HEK293 cells with the two heavy and light chaingene-containing plasmids, cells were grown on selective media asindicated in material and methods. Approximately 40% of the wells showedcell growth after 10 days in culture. Supernatants of clones growing onmedium containing selection markers were tested for IgG secretion byDot-Blot (FIG. 2A), and determination of heavy and light chainproduction using Western-Blot (FIG. 2B). Approximately 90% of the wellswith selected cells were positive for IgG production, with secretionlevels of chimeric IgG between 0.1-20 μg/ml. IgG producing clones wereanalyzed in ELISA for antibody binding to MUC1 (FIG. 2C), showing asignificant correlation between antibody secretion level and theintensity of MUC 1 binding. Positive clone B2 that secreted at 20 μg/ml(FIG. 2D) was selected for further evaluation of chimeric H23 IgG.

A similar procedure was carried out to obtain chFRP5 IgG, where theclone chosen was G1.

Example 2 Expression and Purification of Chimeric IgG1 Derivatives

Chimeric H23 and FRP5 IgG antibodies were purified from stable 293 celllines B2 and G1, respectively, grown in FCS stepwise starvation media asdescribed in Materials and Methods. Stepwise starvation of the cells toFCS not only minimized the contamination with bovine IgG uponpurification on protein A column, but also increased the amount ofantibody secreted to the medium by the FCS deprived cells. Under theseconditions, more than 95% pure chimeric IgG1 protein was obtained basedupon separation on 12%/SDS-PAGE under reducing conditions andverification of human Ig heavy and light chain production byWestern-Blot (FIG. 3). Anti-MUC1 chH23 and anti ErbB2 chFRP5 IgG1antibodies were purified at a total yield of 20 mg per liter of culture.

Example 3 Analysis of chH23 IgG1 Apparent Binding Affinity to MUC1

To compare the binding affinities of chH23 IgG1 with that of H23 mAb, weperformed a comparative half-maximal binding assay using ELISA. Theresults indicated that purified chH23 IgG1 binds MUC1 similarly to theparental mAb. Determination of the apparent binding affinities K_(D) ofthe two antibodies from the half-maximal binding signal revealed thatboth antibodies bind MUC1 with similar apparent affinities, 0.2 nM forthe murine mAb and 0.3 nM for chH23 (FIG. 4). These results suggest thatindeed conversion of the antibody from a monovalent format (Mazor etal., 2005 supra) to a bivalent format as the chimeric H23 is, increasedthe antibody's apparent binding affinity more then a hundred-fold, to alevel roughly equal to that of the parental H23 mAb. This is notsurprising, considering the repetitive nature of the MUC1 antigen thatallows for avidity of the bivalent IgG that are directed to the VNTRregion to be manifested (Hendrikx et al 2002, Am J Pathol 160,1597-608).

To further compare the binding affinities between chH23 and murine H23mAb we tested cellular MUC1 binding by whole-cell ELISA. Cells used inthis assay were the human breast carcinoma T47D cell line. As with thestandard ELISA results, the results of this assay showed that bothantibodies bind the MUC1 positive tumor cells with similar affinity,0.25 nM for the murine mAb and 0.3 nM for chH23 (FIG. 5A). Furthermore,this study demonstrate the specificity of the two antibodies to cellularMUC1, as binding of the MUC1-specific antibodies to the cells could becompeted off with an excess of soluble MUC1 protein.

When evaluated for specific cellular-ErbB2 binding, chFRP5 IgG1exhibited similar binding characteristics as the commercial FDA-approvedanti-ErbB2 Mab Herceptin® (Trastuzumab) (FIG. 5B). Estimation of theapparent binding affinities of the two anti-ErbB2 antibodies from thehalf-maximal binding signal of this whole-cell ELISA on SKBR3 cellsrevealed that both antibodies bind ErbB2 receptor with similar apparentaffinities, 1.7 nM for chFRP5 IgG1 and 2.4 nM for Herceptin®.

Example 4 Comparative Flow Cytometry Analysis

We further evaluated the binding capabilities of the chimeric and murineH23 antibodies by testing cellular MUC1 binding using flow cytometry.Cell lines used in this assay were the human breast carcinoma lines T47Dand MCF-7, the mouse cell line DA3, the MUC1-transfected cell lineDA3-MUC1 (Baruch et al., 1999), and the control human kidney cell lineHEK293. FIG. 6A) indicates that chH23 stains the breast cancer celllines and the MUC1 transfected cells with substantially the same patternas the murine H23 mAb. T47D cells express higher levels of MUC1 comparedto MCF-7 cells and with a heterogeneous population of MUC1 displayingcells in comparison to the homogeneous MUC1-DA3 cells, whereas nobinding was seen with the control DA3 and HEK293 cell lines.Furthermore, we demonstrated the specificity of the two antibodies tocellular MUC1 was demonstrated, as binding of the both antibodies toMUC1 positive cells was competed off following incubation of theantibodies with an excess of MUC1 conditioned medium prior to incubationwith the cells.

Comprehensive flow cytometry analysis on a large set of tumor cellsexpressing varying levels of ErbB2 antigen revealed that chFRP5recognized cellular ErbB2 and stains the tumor cell lines withsubstantially the same pattern as does the anti-ErbB2 Herceptin® Mab(FIG. 6B). The differences in staining intensities between the varioustumor cells usually corresponded to the ErbB2 expression level among theindividual tumor cell lines.

Example 5 Expression and Purification of ZZ-PE38 Fusion Protein

Periplasmic production of soluble ZZ-PE38 fusion was performed asdescribed in Materials and Methods. Periplasmic fractions were preparedand ZZ-PE38 fusion protein was purified by two sequential chromatographysteps of Q-SEPHAROSE and MONO-Q anion exchange columns using fastprotein liquid chromatography (FPLC) until >90% purification wasachieved as seen in FIG. 8A.

Example 6 Preparation of chH23-ZZ-PE38 Immunoconjugate

Conjugation of purified chH23 IgG1 antibody with ZZ-PE38 fusion proteinwas performed as described in Materials and Methods. Separation ofexcess ZZ-PE38 and unconjugated mAb from the chH23-ZZ-PE38 complex wasperformed by applying the sample to a SUPERDEX 75 size-exclusion columnusing fast protein liquid chromatography (FPLC). Purified chH23-ZZ-PE38immunoconjugate was indicated by 12%/SDS-PAGE under reducing conditions(FIG. 8B).

Example 7 Binding Properties of the chH23-ZZ-PE38 Immunoconjugate

Whole-Cell ELISA

To evaluate the binding capabilities of chH23-ZZ-PE38 immunotoxin weperformed cellular MUC1 binding assay by whole-cell ELISA using thehuman breast carcinoma T47D cell line. Rabbit anti-PE sera mixed withHRP-conjugated goat anti-rabbit were used for the detection of boundchH23-ZZ-PE38 immunotoxin. The results of this assay showed that theaffinity tagging of the ZZ-PE38 fusion protein to chH23 Fc domain wasstable, furthermore, the conjugation process did not undermine chH23antigen recognition as the immunoconjugate retained its bindingcapabilities to MUC1 positive tumor cells with comparable affinity tothe un-conjugated chH23 IgG1 (FIG. 9A).

Flow Cytometry Analysis

The binding capabilities of the chH23-ZZ-PE38 immunoconjugate by testingcellular MUC1 binding was further evaluated using flow cytometry. FIG.9B indicates that chH23-ZZ-PE38 stains the MUC1 positive human breastcarcinoma T47D cells while the control human IgG-ZZ-PE38 immunoconjugateprepared and purified in the same way showed no binding to the tumorcells. Furthermore, the specificity of chH23-ZZ-PE38 immunoconjugate tocellular MUC1 was demonstrated, as binding of the anti-MUC1 immunotoxinto the tumor cells was competed off with the addition of 10-fold excessof un-conjugated chH23 IgG1.

Example 8 Cytotoxic Activity of chH23-ZZ-PE38

To evaluate the cytotoxic activity of chH23-ZZ-PE38 immunotoxin, invitro cell-killing experiments were performed. T47D and MCF7 humanbreast carcinoma cell lines were incubated for 48 hr with varyingconcentrations of chH23-ZZ-PE38 and of the control proteins. Therelative number of viable cells in comparison with cells grown in theabsence of toxin was determined using an enzymatic MTT assay. Theresults of the MTT assay (FIG. 10) show that both the highlyMUC1-expressing T47D cells (panel A) and the moderate MUC 1-expressingMCF7 cells (panel B) showed almost no sensitivity to chH23-ZZ-PE38immunconjugate-mediated killing, with an IC₅₀ of ˜5 μg/ml for T47D cellsand undetectable sensitivity for MCF7 cells to the concentrations ofchH23-ZZ-PE38 used in this assay. Comparable results were obtained withthe negative control hIgG-ZZ-PE38 onjugate. These results indicate thatchH23-ZZ-PE38 was not cytotoxic to the tested MUC1-expressing tumorcells; since sensitivity of reported immunotoxin-targeted cellsrepresented IC₅₀ values at the range of 0.1-10 ng/ml (Pai et al 1991,Proc Natl Acad Sci USA 88, 3358-62). These results suggest that chH23may not be suitable for delivering a therapeutic cargo into particulartarget cells i.e. those into which chH23 does not internalize.

Example 9 Preparation of chFRP5-ZZ-PE38 Immunoconjugate

The ZZ-PE38 fusion protein was prepared as described in Materials andMethods. Conjugation of purified chFRP5 IgG1 antibody with the ZZ-PE38fusion protein was performed as described for chH23 above. Separation ofexcess ZZ-PE38 and unlabeled mAb from chFRP5-ZZ-PE38 complex wasperformed by applying the sample on a SUPERDEX 75 size-exclusion columnusing FPLC. The chFRP5-ZZ-PE38 immunoconjugate was obtained as a highlypure immunoconjugate as evident from the SDS-PAGE analysis (FIG. 8C).

Example 10 Binding Analysis of chFRP5-ZZ-PE38 Immunoconjugate

To evaluate the binding capabilities of chFRP5-ZZ-PE38 immunotoxin,cellular ErbB2 binding assay by whole-cell ELISA was performed. Thebinding reactivity of the bivalent chFRP5-ZZ-PE38 immunoconjugate wascompared with that of a recombinant scFv(FRP5)-ETA monovalent derivative(Wels et al., 1995 supra). FIG. 11 shows that chFRP5-ZZ-PE38 retainedits binding activity to cellular ErbB2-expressing SKBR3 tumor cells withsimilar apparent affinity as the un-conjugated form of chFRP5 IgG1. Thisindicated that the affinity tagging of the ZZ-PE38 fusion protein tochFRP5 Fc domain did not undermine chFRP5 antigen recognition. However,comparison of the apparent binding affinities of chFRP5-ZZ-PE38 withthat of the recombinant scFv(FRP5)-ETA by determining the concentrationof immunotoxin that produces half maximal specific binding signalrevealed that the bivalent immunotoxin had an apparent affinity of 1.7nM, whereas the monovalent immunotoxin exhibited a 20-fold lowerapparent affinity of 30 nM. This result is in agreement with theaffinities values reported for scFv(FRP5)-ETA and the parental FRP5 Mab(Wels et al., 1992 supra). The binding activities of both forms of FRP5immunotoxins were further compared by flow cytometry analysis usingequal molar concentration of the two immunotoxins against a large set oftumor cells expressing varying levels of ErbB2 antigen. The results(FIG. 12) show that with all tumors cell lines tested, the bivalentchFRP5-ZZ-PE38 (A) produced more intense staining then the counterpartrecombinant scFv(FRP5)-ETA monovalent derivative (B) as shown by theoverlapping staining intensities results obtained by both immunotoxins(C). These results correlate with the binding affinities obtained forthe two immunotoxins by whole-cell ELISA and emphasize the potentialsuperiority of a bivalent antibody over its monovalent scFv derivative.Furthermore, the specificity of chFRP5-ZZ-PE38 immunoconjugate bindingto cellular ErbB2 was demonstrated, as binding of the bivalentimmunotoxin to the SKBR3 tumor cells could be competed off with theaddition of 10-fold excess of un-conjugated chFRP5 IgG1 (FIG. 13A). Thecontrol human IgG-ZZ-PE38 immunoconjugate prepared and purified in thesame way showed no binding to the SKBR3 tumor cells (FIG. 13B).

Example 11 Cell-Killing Activities of chFRP5-ZZ-PE38

To evaluate the cytotoxic activity of chFRP5-ZZ-PE38 immunoconjugate, weperformed cell-killing experiments on a large set of human tumor celllines expressing varying levels of ErbB2 receptors as shown in Table 2.Cells were incubated for 48 hr with various concentrations ofchFRP5-ZZ-PE38 and the corresponding control proteins and the relativenumber of viable cells was determined using an enzymatic MTT assay. Theresults are shown in FIG. 14 and the IC₅₀ values are summarized in Table2. The results indicated that chFRP5-ZZ-P38 was cytotoxic for all fivecell lines tested with IC₅₀ values that in most cases correlate with thelevels of ErbB2 expression among the different cells. SKBR3 and A431tumor cells were most sensitive to the immunoconjugate with IC₅₀ valuesof 3.5 ng/ml and 1.8 ng/ml respectively. T47D and MCF7 cells weremoderately sensitive to chFRP5-ZZ-PE38 while MDA-MB231 cells showed verylow sensitivity which in some cases reflects a lower level of ErbB2expression of approximately 10³ or less receptors per cell. Takentogether, the cell killing activities achieved with chFRP5-ZZ-PE38 are aconsequence of specific targeting and internalization of theimmunoconjugate into the tumor cells, since none of the separablecomponents alone nor the negative control hIgG-ZZ-PE38 weresignificantly toxic towards any of the tumor cells. The cytotoxicactivities of the chFRP5-ZZ-PE38 immunoconjugate toward A431 cellscorrelated to the IC₅₀ values reported for the anti-mucin B1 mAbconjugated chemically to a truncated LysPE40 derivative of Pseudomonasexotoxin A (Pai et al 1991, Proc Natl Acad Sci USA 88, 3358-62) andexceeded the results reported for the anti-mesothelin K1 mAb conjugatedchemically to a truncated LysPE38QQR derivative of Pseudomonas exotoxinA (Hassan et al 2000, J Immunother 23, 473-9).

TABLE 2 In vitro cell-killing activity Number of ErbB2 ChFRP5- IC₅₀(ng/ml) ZZ- Cell line receptors ZZ-PE38 hIgG-ZZ-PE38 PE38 ChFRP5 SKBR31.5 × 10⁶ 3.5 >10000 >10000 >10000 A431   2 × 10⁴ 1.8 3500 >10000 >10000T47D   ~10⁴ 1000 >10000 >10000 >10000 MCF7 <1000600 >10000 >10000 >10000 MDA- <1000 >10000 >10000 >10000 >10000 MB231

Comparison of the cytotoxic activities of the bivalent chFRP5-ZZ-PE38with that of the monovalent scFv(FRP5)-ETA using equal molarconcentration of each immunotoxin showed that the former was much morepotent than the latter, with IC₅₀ values of 30 pM vs 1000 pMrespectively (in SKBR3 cells), and 10 pM vs 52 pM respectively (in A431cells; see FIG. 15 and Table 3). Moreover, while the lowErbB2-expressing MCF7 and MDA-MB231 cells showed moderate sensitivity tochFRP5-ZZ-PE38, these cells were totally unaffected by equimolarconcentrations of the monovalent scFv(FRP5)-ETA. The specificity of thetwo immunotoxins in targeting and killing ErbB2-expressing tumor cellswas demonstrated, since the cytotoxic activity of both immunotoxins wascompeted off in the presence of excess chFRP5 IgG1. Our experimentalIC₅₀ values achieved for the monovalent scFv(FRP5)-ETA are in fullagreement with the results obtained with this immunotoxin by (Wels et al1992, supra; Wels et al 1995, supra). We undoubtedly demonstrated thatreformatting the monovalent scFv immunotoxin into a bivalent IgGimproved dramatically the cytotoxic activities of this anti-ErbB2immunotoxin. Taken together, the cytotoxic activity of both immunotoxinscorrelates with the binding affinities obtained for the two immunotoxinsby the whole-cell ELISA (FIG. 11) and matches the cellular ErbB2 bindingactivities obtained by flow cytometry (FIG. 12) against the same set oftumor cell lines. This further emphasizes the superiority of thebivalent chFRP5 IgG1 antibody over its monovalent scFv derivative. Thissuperiority is mainly important when evaluating an immunotoxin fortherapeutic application as the affinity of the antibody counterpart is amajor determining factor in establishing how fast it will bind to atumor cell and how quickly it will released from the antigen-bearingtumor cell (Henderikx et al 2002, supra). Since the residence time oncewithin the tumor mass is suggested to be controlled by affinity andavidity (Milenic et al 1991, Cancer Res 51 (23 Pt 1:6363-71). bivalentantibodies may be advantageous.

TABLE 3 In vitro cell-killing activity of chFRP5-ZZ-PE38 andscFv(FRP5)-ETA ChFRP5- Number of IC₅₀ (pM) ZZ- scFv(FRP5)- Cell ErbB2ChFRP5- scFv(FRP5)- PE38 + ETA + line receptors ZZ-PE38 ETA chFRP5chFRP5 SKBR3 1.5 × 10⁶ 30 1000 10000 >100000 A431   2 × 10⁴ 10 5210000 >100000 MCF7 <1000 300 >10000 >100000 >100000 MDA- <100030000 >100000 >100000 >100000 MB231

Example 12 Functional Stability Analysis of chFRP5-ZZ-PE38Immunoconjugate Complex

In order to estimate the stability of the ZZ-Fc affinity connectionwithin the chFRP5-ZZ-PE38 complex we incubated the purifiedimmunoconjugate at 37° C. for varying periods in the presence of 10-foldmolar excess of protein-A purified human IgG as competitor before it wastested for cellular-ErbB2 binding of SKBR3 cells by whole-cell ELISA.The results (FIG. 17A) show that when incubated in the presence of humanIgG competitors, chFRP5-ZZ-PE38 immunoconjugate lost more then 80% ofits binding activities after 24 hr as compared to untreatedchFRP5-ZZ-PE38 that retain full binding activities even after 7 days at37° C. However, since commercial protein-A purified human IgG antibodiesdo not represent an accurate state of protein A-binding immunoglobulinspresent in human serum, we also estimated the functional stability ofthe immunoconjugate complex subsequent to incubation at 37° C. for 72 hin 100% human serum from three individual healthy donors before testedfor cellular-ErbB2 binding of SKBR3 cells. The results (FIG. 17B) showthat with all three human sera tested; chFRP5-ZZ-PE38 immunoconjugatelost only 10-30% of its binding activities as compared to theimmunoconjugate complex that was incubated in PBS. The modest loss ofactivity obtained in this assay compared with the drastic loss ofactivity seen after pre-incubation of the immunoconjugate in thepresence of protein-A purified human IgG antibodies, represents a moregenuine condition in which the functional stability of chFRP5-ZZ-PE38immunoconjugate should be tested since it is mimicking the conditions inwhich the functional stability of the chFRP5-ZZ-PE38 immunoconjugatewill encounter in a live animal model. In particular, not all humanserum immunoglobulins have the capability of affinity binding to ZZdomain, while commercial human IgG antibodies as used for thecompetition assay are routinely all protein-A purified, and thusrepresent an enriched population of high-affinity ZZ domain binders.

Example 13 Construction of Crosslinked chFRP5-ZZ-PE38 Immunoconjugate

To circumvent the undesirable event in which the affinity-interactionbetween chFRP5 Fc domain and the ZZ-PE38 component could be competed offpost-formation by protein-A-binding immunoglobulin orimmunoglobulin-like competitors, a crosslinked derivative of thechFRP5-ZZ-PE38 immunoconjugate complex was prepared using thecross-linking reagent BS³ (Pierce, USA). Following the crosslinkingreaction, more then 80% of the immunoconjugate was covalentlycrosslinked as indicated by SDS-PAGE analysis (FIG. 18A). Thecrosslinked derivative had a total size of about 200 kDa consisting ofcovalently linked heavy and light chains of chFRP5 IgG1 (150 kDa)together with the 50 kDa ZZ-PE38 component. When evaluated for antigenbinding, the crosslinked-chFRP5-ZZ-PE38 retained the same apparentaffinities to cellular-ErbB2 of human SKBR3 tumor cells as thenon-crosslinked derivative (FIG. 18B). This confirmed that thecrosslinking process did not undermine the binding activity of theimmunoconjugate. When tested for functional stability followedincubation for 24 h at 37° C. in the presence of 10-fold molar excess ofthe ZZ-binding human IgG1 antibody as competitor, the un-conjugatedchFRP5-ZZ-PE38 lost more then 80% of its binding activity, while thecrosslinked derivative lost only 21% of its binding capability (FIG.18B). This loss of activity probably corresponds to the fraction ofchFRP5-ZZ-PE38 that was not covalently-linked during the crosslinkingprocess i.e. about 80% efficiency in crosslinking was observed.Furthermore, incubation of the crosslinked-immunoconjugate in 100% humanserum did not result in any loss of binding activity compared with adecrease of about 17% for the non-crosslinked derivative.

We further evaluated the functional stability and cytotoxic activitiesof the crosslinked-chFRP5-ZZ-PE38 complex by analyzing cell-killingactivities of the modified immunoconjugate following 24 h incubation at37° C. with protein-A purified human IgG antibodies (FIG. 19A) or in thepresence of 100% human sera (FIG. 19B). While the crosslinking processdid not undermine the binding affinity of the covalent-linkedimmunoconjugate, the cell-killing assays demonstrated that there wassome reduction of in the cytotoxic activity of thecrosslinked-chFRP5-ZZ-PE38 as indicated by IC₅₀ values of 1.8 ng/ml vs 6ng/ml (Table 4). However, while the non-crosslinked derivative wastotally non-toxic to A431 cells following incubation with human IgG1competitors (IC₅₀ of >1000 ng/ml), the crosslinked-immunoconjugate onthe other hand retained most of its cytotoxic activity with IC₅₀ valuesof 11 ng/ml. As with the whole-cell ELISA results (FIG. 19B), incubationof the crosslinked-immunoconjugate in 100% human serum did not result inany loss of cytotoxic activity while moderate reduction in cell-killingactivities was observed for the non-crosslinked derivative with IC₅₀values of 2.7 ng/ml. Taken together, the results show that crosslinkingof chFRP5-ZZ-PE38 does not affect its binding affinities and furthersignificantly improves its functional stability as compared to thenon-crosslinked derivative. However crosslinking may lead to a reductionin the cytotoxic activity of the immunoconjugate, suggesting that “finetuning” of the crosslinking conditions is required to fully maintain thecytotoxic activity. The observed loss in cytotoxicity may result todamage to the toxin moiety. A derivative of PE38, namely PE38-QQR hasbeen reported to be resistant to chemical-conjugation induced damage(Debinski and Pastan 1994, Bioconjug Chem 5, 40-6).

TABLE 4 In vitro cell killing activities of crosslinked-chFRP5-ZZ-PE38IC₅₀ (ng/ml) Crosslinked Crosslinked ChFRP5- ChFRP5- chFRP5-ZZ-chFRP5-ZZ- Number of ZZ-PE38 + ZZ-PE38 + Crosslinked PE38 + PE38 + ErbB2ChFRP5- Commercial Human chFRP5-ZZ- Commercial Human Cell line receptorsZZ-PE38 hIgG1 serum PE38 hIgG1 serum A431 2 × 10⁴ 1.8 >1000 2.7 6 11 6

Example 14 In Vivo Characterization of chFRP5-ZZ-PE38 in Animal Models

To evaluate the in vivo behavior of the chFRP5-ZZ-PE38 immunoconjugateand also evaluated its potential to serve as a therapeutic agent forspecific targeting and killing of human tumor cells expressing highlevel of ErbB2 receptors, a series of in vivo assays was conducted toanalyze its toxicity and its pharmacokinetic behavior in BALB/c mice. Wefurther evaluated the anti tumor activity of chFRP5-ZZ-PE38 in specifictargeting and killing of tumor xenografts in female BALB/c athymic nudemice. In these studies we compared chFRP5-ZZ-PE38 to the monovalentscFv(FRP5)-ETA immunotoxin.

Example 15 Comparative Pharmacokinetics of chFRP5-ZZ-PE38 andscFv(FRP5)-ETA in Mouse Serum

The pharmacokinetic behavior of chFRP5-ZZ-PE38 and scFv(FRP5)-ETA inBALB/c mice was determined by measuring the immunotoxin levels in bloodsamples drawn from the mice at various time points following i.v.injection of each immunotoxin. Briefly, female BALB/c mice were given asingle i.v. dose of 15 μg chFRP5-ZZ-PE38 or 5 μg scFv(FRP5)-ETA(equimolar dose) by injection into the tail vein. Blood samples weredrawn from the orbital vein of mice injected with scFv(FRP5)-ETA at 2,5, 10, 20, 30, 60, 120 and 240 min after injection and at 2, 5, 10, 20,30, 60, 120, 240, 480 and 1440 min for mice injected withchFRP5-ZZ-PE38. The immunotoxin levels in the samples was estimated bydot-blot analysis of serum dilutions alongside a two-fold dilutionseries of chFRP5-ZZ-PE38 (FIG. 20A) or scFv(FRP5)-ETA (FIG. 20B)standards, the concentration of remaining active immunotoxin wasdetermined by incubating dilutions of the serum with A431 cells andmeasuring cell-killing activity by MTT assay. The pharmacokineticscurves of chFRP5-ZZ-PE38 and scFv(FRP5)-ETA were determined bycalculating the results of both the dot-blot and MTT assays and arepresented in FIG. 21. The results obtained show that while thechFRP5-ZZ-PE38 immunoconjugate has a serum half-life of 240 min thatcorrelated to the t1/2 of different IgG-Pseudomonas exotoxin Aimmunoconjugates (Pai et al 1991, Proc Natl Acad Sci USA 88, 3358-62),the recombinant scFv(FRP5)-ETA immunotoxin half-life of only 18 min,similar to previous results obtained for this immunotoxin (Wels et al1992, Cancer Res 52, 6310-7) and was in agreement to the t1/2 of otherFv immunotoxins of similar size (Reiter et al 1994, Biochemistry 33,5451-9; Benhar and Pastan 1995, Clin Cancer Res 1, 1023-1029).

In vitro experiments show that once distributed into the readilyaccessible extracellular space, the anti tumor efficacy of animmunotoxin is a function of its affinity for the tumor cells and itsability to penetrate into the tumor (Benhar and Pastan, 1995, supra),thus the antibody fragment must stay in the circulation long enough todiffuse from the blood stream to the tumor without being cleared by thekidneys. It was calculated that it should take about 5-6 h for animmunotoxin to equilibrate within the tumors (Benhar and Pastan, 1995supra). Our pharmacokinetic results predict a considerable advantage forchFRP5-ZZ-PE38 in its anti tumor activity over the scFv(FRP5)-ETAderivative, since the t1/2 of the latter is only 18 min, there should besignificant clearance of scFv(FRP5)-ETA immunotoxin molecules duringthis period, whereas very small fraction of chFRP5-ZZ-PE38immunoconjugate would be cleared at the same period of time.

Example 16 Toxicity of ZZ-PE38 and ZZ-PE38 Immunoconjugates in Mice

The single-dose mouse LD₅₀ of ZZ-PE38 fusion protein and threeimmunoconjugates; chFRP5-ZZ-PE38, crosslinked-chFRP5-ZZ-PE38 andhIgG-ZZ-PE38 was evaluated in female BALB/c mice that were given asingle i.v. injection with different doses of each protein. All micewere monitored for weight loss or death for 14 days post-injection. Theresults of the toxicity assay (Table 5) show that there is a significantdifference in toxicity between the chFRP5-ZZ-PE38 and human IgG-ZZ-PE38immunoconjugates as indicated by LD₅₀ of 0.75-1 mg/kg vs 2.5 mg/kgrespectively. This difference in toxicity can be the consequence of aweaker affinity conjugation of the ZZ-PE38 component to the Fc domain ofchFRP5 IgG1 which can lead to unspecific binding of the ZZ-PE38 fusionprotein to endogenous mouse immunoglobulins that have stronger affinityto the ZZ-PE38 component. In contrast, covalent-crosslinking of thechFRP5-ZZ-PE38 abrogated the excessive toxicity as mice were not killedfollowing injection with 1.25 mg/kg of the crosslinked-chFRP5-ZZ-PE38derivative up to 14 days post-injection. However, when ZZ-PE38 wasinjected to mice it was significantly toxic with an LD₅₀ of 0.375 mg/kg.This highlights the need for optimization of a crosslinking method, oralternative linking methods that preserve the integrity of theIgG-ZZ-PE38 complex and its full cytotoxic potency.

Example 17 Anti Tumor Activity of chFRP5-ZZ-PE38 Immunotoxin in NudeMice

To study the in vivo activity of chFRP5-ZZ-PE38 immunoconjugate, weevaluated its anti tumor activity in the eradication of A431 tumorxenografts in athymic nude mice. We further compared the therapeuticpotential of the bivalent immunoconjugate chFRP5-ZZ-PE38 with that ofthe monovalent scFv(FRP5)-ETA. Tumors were induced in athymic nude miceby s.c. injection of 1.5×10⁶ A431 cells on day 0. Treatment wasinitiated on day 9 when the tumors averaged 30-40 mm³ in volume, andconsisted of five i.v. injections on days 9, 12, 15, 18, and 21 ofvarious doses of the immunotoxins. Control mice were treated with PBSonly. As shown in FIGS. 22 and 23, chFRP5-ZZ-PE38 exhibited asignificant antitumor effect. Mice treated at a dose of 0.5 mg/kg (FIG.23D) exhibited complete remission of the tumor that lasted for twomonths until the animals were sacrificed, and mice treated at a dose of0.25 mg/kg (FIG. 23C), exhibited arrested tumor growth, in most casesfor the duration of the experiment i.e. until the animals weresacrificed after two months. In some animals, tumor growth resumed a fewweeks after treatment.

In contrast, treatment of mice with 0.25 mg/kg of the monovalentscFv(FRP5)-ETA (FIG. 23B), a dose which correlates to 1.5 and 3 foldhigher molar concentration as compared to the doses used with thebivalent immunoconjugate, resulted in minimal to no effect on tumorgrowth. Tumor size in control mice treated with PBS (FIG. 23A) increasedprogressively. By day 30, tumor size in control and scFv(FRP5)-ETAtreated animals was about 1.0 cm³. When the experiment was terminated onday 30 by euthanization of the animals, the size of the tumors in thescFv(FRP5)-ETA-treated animals was 96% that of the tumor size in thecontrol group as compared to 5.5% and 19% for animals treated with 0.5and 0.25 mg/kg chFRP5-ZZ-PE38 respectively. No drug-induced toxicity inmice was observed at the doses of scFv(FRP5)-ETA and chFRP5-ZZ-PE38 usedin this study.

The less than optimal anti tumor effects obtained with scFv(FRP5)-ETA ata dose of 0.25 mg/kg was not surprising as it was previously reportedthat A431 xenografts bearing athymic nude mice receiving twice-dailyi.p. injections of 0.25 mg/kg of scFv(FRP5)-ETA for a total of 10 daysled only to a modest inhibition of tumor growth, yielding tumors 44% ofthe size of the control group (Wels et al 1995, Int J Cancer 60,137-44). Our results demonstrate the superiority of the bivalentimmunoconjugate over its monovalent counterpart in targeted cancertherapy of live athymic nude mice bearing A431 tumor xenografts.

Example 18 Preparation of Cetuximab—ZZ-PE38 Immunotoxin Complex

ZZ-PE38 was prepared using E. coli strain BL21 (DE3) transformed by theplasmid pET22-NN-ZZ-PE38. Fusion protein—ZZ-PE38 is arranged from two Zdomains of protein A produced by Staphylococcus aureus and from 38 kDafragment of exotoxin A produced by Pseudomonas aeruginosa. Pseudomonasexotoxin is capable of killing mammalian cells by inhibiting proteinsynthesis.

The products obtained at each stage of preparing immunotoxin complexwere analyzed using SDS-PAGE gel stained by Coomassie blue. The resultspresented in lanes 1 and 2 of FIG. 24 demonstrate successfulpurification and concentration of ZZ-PE38 protein. The incubation ofcetuximab with ZZ-PE38 obtained at molar ratio of 1:3 over nightresulted in the formation of non-covalently bound complex (FIG. 24, lane4). The immunotoxin complex was separated from unbound ZZ-PE38 bySUPERDEX 75 gel filtration, dialyzed with PBS and then concentrated to2-3 mg/ml using centrifugal filter with 100 kD cutoff.

In addition, the covalently bound, cross-linked cetuximab-ZZ-PE38immunotoxin complex was obtained using BS3 cross linker (FIG. 24, lane5). This complex was found to be stable in the presence of SDS whennon-covalently bound immunotoxin complex dissociated to antibody (˜150kDa) and ZZ-PE38 (>50 kDa).

Example 19 Effect of Cetuximab—ZZ-PE38 Complex on Survival of CancerCells

The efficacy of the obtained immunotoxin complex was tested on H&N andprostate cancer cell lines as well as on human normal fibroblasts. Thesecells were characterized by different expression of ErbB-1 (FIGS. 25 and27): high expression of ErbB-1 was revealed by Western blot assay andconfirmed by ELISA, confocal microscopy and flow cytometry in SCC-9 H&Ncancer cells and in C1-1 clone 3 prostate cancer cells while theexpression of ErbB-1 in SCC-1 and C1-1 clone 7 cells was lower. ErbB-1was not detectable in human normal fibroblasts (Western blot assay) andcetuximab binding to these cells was very low and non-specific.

The inhibitory effect of immunotoxin complex on the survival of ErbB-1positive SCC-1 cells was compared to the efficacy of unbound ZZ-PE38 andnon specific human IgG-ZZ-PE38 complex (FIG. 26). The immunotoxincomplex (IC₅₀=0.00047 μg/ml) was two orders of magnitude more effectivein inhibiting the growth of SCC-1 cells than IgG-ZZ-PE38 complex(IC₅₀=0.036 μg/ml) and four orders of magnitude more effective thenZZ-PE38 alone (IC₅₀=2.51 μg/ml) (p<0.0001 for both treatments).

The efficacy of the immunotoxin complex was found to be in correlationwith the level of ErbB-1 expression in the cells. As shown in FIG. 27,the sensitivity of ErbB-1 highly positive SCC-9 (IC50=0.000073 μg/ml)cells to immunotoxin complex was significantly higher (p<0.0001) thanthe sensitivity of SCC-1 cells (IC₅₀=0.000261 μg/ml). Similar data werereceived for C1-1 prostate cancer cells (FIG. 27): clone 3 cells withhigher ErbB-1 level were more sensitive (IC₅₀=0.012 μg/ml) than clone 7cells (IC₅₀=0.038 μg/ml) (p<0.0001).

This statement was supported by the results obtained from normal humanforeskin fibroblasts. These non-cancerous cells did not expressmeasurable quantity of ErbB-1 (tested by Western blot analysis—data notshown) and the binding of cetuximab to these cells was low andunspecific (FIG. 27). At low concentration of immunotoxin complex (0.001μg/ml that killed 80% of SCC-1 cells) no damage to human fibroblasts wasnoted (FIG. 27). At the maximal concentration of immunotoxin complex (30μg/ml), the survival of SCC-1 cells was <10% compared to 60% of humanfibroblasts (p<0.0001).

In addition, the efficacy of the immunotoxin complex was tested in thepresence and the absence of non-specific human IgG (in the concentration3 times higher than the concentration of the complex) to imitate theconditions of complex injection (patient treatment). As shown in FIG.28, the presence of IgG did not change complex efficacy. It was alsofound that the non-cross linked immunotoxin complex was more effective(IC50=0.00031 μg/ml) than the more stable cross linked form(IC50=0.00754 μg/ml) (p<0.0001) (FIG. 28).

Example 20 Effect of Cetuximab—ZZ-PE38 Complex on Tumor Growth

In preliminary experiments, a toxicity of immunocomplex at 0.350 and0.500 μg/g was evaluated in intact Balb/c mice. 10 mice were used foreach group. In the group treated by low concentration of the complex,none of the mice died. At higher dose of the complex the mice started todie after 24 hrs (15% died). After 72 hrs 75% of the mice died. The lastmouse died at 94 and 120 hrs. Pathology analysis revealed that livertoxicity was associated with death.

Subcutaneous implantation of SCC-1 human cancer cells generated fastgrowing tumors in nude mice. Tumor volume was calculated twice a weekand was based on the measurement of tumor size in two perpendiculardirections. Two weeks following cell implantation the mice wererandomized by the tumor size in 5 groups (5 mice overall bearing 8-10tumors in each group): control mice (group 1), mice treated by ZZ-PE38(0.20 μg/g) alone (group 2), mice treated by cetuximab (0.40 μg/g) alone(group 3), mice treated by cetuximab-ZZ-PE38 complex (0.25 μg/g) (group4), and mice treated by cetuximab-ZZ-PE38 complex (0.50 μg/g) (group 5).All agents tested were administrated intraperitoneally twice a week forthree weeks.

In mice untreated and treated by ZZ-PE38 alone, tumor growth was fastand similar: the tumor volume increased from ˜70 mm³ at baseline to ˜300mm³ at the end of the experiment (FIG. 29). Cetuximab alonesignificantly decreased tumor growth relative to untreated mice(p<0.042). In this group of mice the tumor size increased from ˜80 mm³at the beginning of the treatment to ˜200 mm³ at the end of theexperiment. The treatment with cetuximab-ZZ-PE38 complex at low and highconcentrations resulted in a significant tumor shrinkage (FIG. 29). Theimmunocomplex at the lower dose was significantly more effective thancetuximab alone (p<0.01). As expected, immunotoxin complex at the higherdose was more effective then at the lower dose (FIG. 29A) but thedifference between these two groups was not statistically significant(p<0.06). Tumor growth resumed a week after completion of immunotoxinadministration. It has to be noted that 3 mice out of the 5 tumorbearing mice injected with the high dose (0.500 mg/Kg) of cetuximab-ZZPE38 complex died the day after the first injection. Two surviving micefrom this group as well as all mice treated by low dose of the complexdid not suffer from side effects during follow up. In the dead miceliver toxicity was observed.

These results surprisingly provide that Cetuximab, a chimericanti-ErbB-1 antibody, known to possess limited efficacy in prostateneoplasm, can be synergistically combined with a fusion proteinpossessing cytotoxic activity as described herein. Thus the immunotoxinprovides means of introducing the fusion protein in sub-toxic-tolerabledosages that are still effective in treating cancer.

Specifically, this study we demonstrated the efficacy of a novelanticancer immunotoxin—cetuximab combined with ZZ-PE38 both in in vitroand in vivo experiments using H&N and prostate cancer cells expressingdifferent levels of ErbB-1. It is important to emphasize that theimmunotoxin utilized was found to be more effective then the mere use ofeither ZZ-PE38 alone or ZZ-PE38 combined with non specific IgG in bothtypes of cancer cells tested. Moreover, the enhanced cytotoxic effectwas higher in those cell lines/clones that have higher level of ErbB-1.The cytotoxic effect of cetuximab-ZZ-PE38 immunotoxin on normalfibroblast cells, characterized by non measurable level of ErbB-1, wasfound only at very high dosage. Thus the present invention provides thatcetuximab-ZZ-PE38 exhibits an ErbB-1 specific activity. This phenomenonrenders this composition safe and tolerable to non-cancerous cells thatexpress law or undetectable levels of ErbB-1.

These novel experiments also show by using an ectopic model of tumorgenerated by SCC-1 cells in nude mice that the immunotoxin at non-toxicdose (0.25 μg/g) induced significant tumor shrinkage while cetuximabalone at a higher dose (0.40 μg/g) only decreased tumor growth rate.Toxin alone at the dose used did not have any anti-tumor effect.

In conclusion, Cetuximab-ZZ-PE38 complex was found to be synergisticallymore effective than cetuximab or ZZ-PE38 alone in human H&N and prostatecancer cells in both in vitro and in vivo studies.

1. An immunotoxin comprising a fusion protein and an anti-ErbB-1antibody, wherein the fusion protein comprises an immunoglobulinFc-binding domain and a truncated form of Pseudomonas exotoxin.
 2. Theimmunotoxin according to claim 1, wherein the antibody is selected formthe group consisting of a monoclonal antibody, a humanized antibody, achimeric antibody, a single chain antibody, and a fragment thereof andis capable of being internalized into the target cell.
 3. Theimmunotoxin according to claim 1, wherein the fusion protein comprisesthe amino acid sequence of SEQ ID NO:1.
 4. The immunotoxin according toclaim 1, wherein the fusion protein and the anti-ErbB-1 antibody arecross linked or non-cross linked.
 5. The immunotoxin according to claim1, wherein the anti-ErbB-1 antibody is Cetuximab.
 6. A compositioncomprising the immunotoxin of claim 1 and a pharmaceutically acceptablecarrier.
 7. A method for treating a subject afflicted with an ErbB-1associated cancer, comprising administering to the subject atherapeutically effective amount of a composition comprising animmunotoxin, the immunotoxin comprises a fusion protein and ananti-ErbB-1 antibody, wherein the fusion protein comprises animmunoglobulin Fc-binding domain and a truncated form of Pseudomonasexotoxin, thereby treating a subject afflicted with an ErbB-1 associatedcancer.
 8. The method of claim 7, wherein the fusion protein comprisesthe amino acid sequence of SEQ ID NO:1.
 9. The method of claim 7,wherein the treating a subject afflicted with an ErbB-1 associatedcancer is inducing cell death in cancer cells expressing ErbB-1.
 10. Themethod of claim 7, wherein the antibody is selected form the groupconsisting of a monoclonal antibody, a humanized antibody, a chimericantibody, a single chain antibody, and a fragment thereof and is capableof being internalized into the target cell.
 11. The method of claim 7,wherein the fusion protein and the anti-ErbB-1 antibody are cross linkedor non-cross linked.
 12. The method of claim 7, wherein the ErbB-1associated cancer is lung cancer, anal cancer, glioblastoma multiforme,epithelial cancer, prostate cancer, pancreatic cancer, head and neckcancer, breast cancer, ovarian cancer, renal cancer, or any combinationthereof.
 13. The method of claim 7, wherein the anti-ErbB-1 antibody isCetuximab.
 14. A method for inducing cell death in a cell expressingErbB-1, comprising the step of contacting the cell with atherapeutically effective amount of a composition comprising animmunotoxin, the immunotoxin comprises a fusion protein and ananti-ErbB-1 antibody, wherein the fusion protein comprises animmunoglobulin Fc-binding domain and a truncated form of Pseudomonasexotoxin, thereby inducing cell death in a cell expressing ErbB-1. 15.The method of claim 14, wherein the cell is a cancer cell.
 16. Themethod of claim 14, wherein the fusion protein comprises the amino acidsequence of SEQ ID NO:1.
 17. The method of claim 14, wherein theantibody is selected form the group consisting of a monoclonal antibody,a humanized antibody, a chimeric antibody, a single chain antibody, anda fragment thereof and is capable of being internalized into the targetcell.
 18. The method of claim 14, wherein the fusion protein and theanti-ErbB-1 antibody are cross linked or non-cross linked.
 19. Themethod of claim 15, wherein the cancer cell expressing ErbB-1 is a lungcancer cell, an anal cancer cell, a glioblastoma multiforme cell, anepithelial cancer cell, a prostate cancer cell, a pancreatic cancercell, a head and neck cancer cell, a breast cancer cell, an ovariancancer cell, or a renal cancer cell.
 20. The method of claim 14, whereinthe anti-ErbB-1 antibody is Cetuximab.